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ISBN 978-951-42-8989-7 (Paperback)ISBN 978-951-42-8990-3 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

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Jukka Leinonen

OULU 2008

D 998

Jukka Leinonen

CARBONIC ANHYDRASE ISOENZYME VI: DISTRIBUTION, CATALYTIC PROPERTIES AND BIOLOGICAL SIGNIFICANCE

FACULTY OF MEDICINE,INSTITUTE OF BIOMEDICINE,DEPARTMENT OF ANATOMY AND CELL BIOLOGY,UNIVERSITY OF OULU;

A C T A U N I V E R S I T A T I S O U L U E N S I SD M e d i c a 9 9 8

JUKKA LEINONEN


Academic dissertation to be presented, with the assent ofthe Faculty of Medicine of the University of Oulu, forpublic defence in Auditorium A101 of the Department ofAnatomy and Cell Biology (Aapistie 7 A), on December19th, 2008, at 12 noon

OULUN YLIOPISTO, OULU 2008

Copyright © 2008Acta Univ. Oul. D 998, 2008

Supervised byProfessor Hannu Rajaniemi

Reviewed byProfessor Jukka H. MeurmanProfessor Pentti Tuohimaa

ISBN 978-951-42-8989-7 (Paperback)ISBN 978-951-42-8990-3 (PDF)http://herkules.oulu.fi/isbn9789514289903/ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)http://herkules.oulu.fi/issn03553221/

Cover designRaimo Ahonen

OULU UNIVERSITY PRESSOULU 2008

Leinonen, Jukka, Carbonic anhydrase isoenzyme VI: distribution, catalyticproperties and biological significanceFaculty of Medicine, Institute of Biomedicine, Department of Anatomy and Cell Biology,University of Oulu, P.O.Box 5000, FI-90014 University of Oulu, Finland Acta Univ. Oul. D 998, 2008Oulu, Finland

AbstractSecretory carbonic anhydrase isoenzyme VI (CA VI) catalyses the reversible hydration of carbondioxide (CO2 + H2O ↔ HCO3

- + H+). Low concentrations of salivary CA VI are associated withhigh decayed, missing or filled teeth (DMFT) index scores and a high incidence of acid injury inthe upper gastrointestinal tract plus lowered taste and smell perception. Two mechanisms of actionfor CA VI have been proposed: acid neutralisation and growth factor function.

In the present study the distribution and catalytic properties of CA VI have been examined inorder to further clarify its mechanisms of action and biological significance. CA VI was found tobe present and secreted by the alveolar epithelium of the mammary gland, serous acinar cells oflingual von Ebner’s glands, serous demilune cells of posterior lingual mucous glands and serouscells of submucosal tracheobronchial glands. CA VI was also found in the serous cells in thetracheobronchial mucosal epithelium, taste pore, taste bud, base of the tracheobronchial cilia,bronchiolar Clara cells and enamel pellicle. An immunofluorometric assay showed that the meanconcentration of CA VI in colostral milk was eight times higher than that in mature milk (35 mg/lvs. 4.5 mg/l). Stopped-flow spectroscopy measurements revealed that the dehydration activity ofCA VI is moderate (maximum kcat = 3.0 × 105 · s-1).

The finding that CA VI is a potent catalyst of acid neutralisation emphasizes the possible roleof the pellicle bound CA VI in local neutralisation of the acidic metabolic products of dentalbiofilm. The function of CA VI in von Ebner’s glands’ saliva is likely taste stimuli modificationvia CA activity although other functions may exist. Its role in milk or respiratory tract mucusremains open, however, as these secretions do not have significant acid predispositions that wouldneed enzymatic catalysis for removal.

Keywords: carbonic anhydrase, catalysis, immunochemistry, milk, saliva, trachea, vonEbner’s glands

To my family

Acknowledgements

This work was carried out at the Department of Anatomy & Cell Biology, Instituteof Biomedicine, University of Oulu. I wish to express my sincere gratitude to mysupervisor Professor Hannu Rajaniemi and to my senior research group membersProfessor Seppo Parkkila and Dr. Kari Kaunisto whose expertise in science andcarbonic anhydrase have been of the utmost importance for the completion of thisthesis.

I am very grateful to Professors Jukka Meurman and Pentti Tuohimaa for theirvaluable comments and constructive criticism on this thesis.

I wish to express my gratitude to my close research buddies Matti Vähäkari,Mika Kaakinen, Jyri Taskila and Harri Palokangas for their collegial support in themany problems a beginning researcher encounters. I would also like to thankProfessor Xilin Ren, Dr. Ulrich Bergmann and Dr. Marja Nissinen for theirgenerous help in the laboratory work of the unpublished data in this thesis plus myco-authors Professor Juha Tapanainen, Dr. Anna-Kaisa Parkkila, Dr. Jyrki Kivelä,Dr. Pepe Karhumaa, Dr. Hanna Myllylä, Dr. Kirsi Saari, Dr. Jaana Seikkula andDr. Petri Koivunen for their support in this work.

In addition, I would like to thank Ms. Lissu Hukkanen, Ms. Paula Soininen,Ms. Marja Paloniemi and Mr. Eero Oja for their technical assistance.

I wish to thank Dr. Deborah Kaska for the revision of the language of themanuscript of this thesis.

Finally I would like to thank my beloved Kaisa, my family, all my friends andall of my co-workers in the Department of Anatomy & Cell Biology, Institute ofBiomedicine plus all the experimental volunteers for their help and patience withmy thesis.

This study was supported by grants from the Finnish Dental AssociationApollonia, the Finnish Cultural Foundation, the Oulu University Grant Foundationand the Emil Aaltonen Foundation.

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Abbreviations

BSA Bovine serum albuminBSA-PBS Bovine serum albumin in phosphate-buffered salineCA Carbonic anhydraseCA VI Carbonic anhydrase isoenzyme VIcDNA Complementary deoxyribonucleic acidDMFT Decayed, missing or filled teeth kcat Turnover number, the maximum number of enzymatic reactions

catalysed per secondKm Michaelis constant, substrate concentration at which the rate of the

enzyme reaction is half of the maximumkDa Kilodalton, unit of molecular massnonO Non-POU (Pit-Oct-Unc) domain-containing octamer-binding proteinp54nrb Nuclear RNA-binding protein, 54 kDaPBS Phosphate-buffered salinepKa Acid dissociation constantPVDF Polyvinylidine fluoridemRNA Messenger ribonucleic acidRT-PCR Reverse-transcriptase-polymerase chain reactionSDS Sodium dodecyl sulphateSDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis

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List of original publications

This thesis is based on the following articles, which are referred to in the text bytheir Roman numerals:

I Leinonen J, Kivelä J, Parkkila S, Parkkila AK & Rajaniemi H (1999) Salivarycarbonic anhydrase isoenzyme VI is located in the human enamel pellicle.Caries Res 33:185–190.

II Leinonen J, Parkkila S, Kaunisto K, Koivunen P & Rajaniemi H (2001)Secretion of carbonic anhydrase isoenzyme VI (CA VI) from human and ratlingual serous von Ebner’s glands. J Histochem Cytochem 49:657–662.

III Karhumaa P, Leinonen J, Parkkila S, Kaunisto K & Rajaniemi H (2001) Theidentification of secreted carbonic anhydrase VI as a constitutive glycoproteinof human and rat milk. Proc Natl Acad Sci USA 98:11604–608.(Karhumaa P and Leinonen J contributed equally to this work)

IV Leinonen J, Saari K, Seppänen J, Myllylä H & Rajaniemi H (2004)Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI(CA VI) expression in rat lower airways and lung. J Histochem Cytochem52:1107–1112.

Additionally, unpublished original data on CA VI are included.

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ContentsAbstractAcknowledgements 7

Abbreviations 9

List of original publications 11

Contents 131 Introduction 152 Review of the literature 17

2.1 Saliva ..................................................................................................... 172.2 Dental caries .......................................................................................... 182.3 Taste perception..................................................................................... 192.4 Milk ....................................................................................................... 212.5 Structure, function and development of the respiratory tract ................ 222.6 Carbonic anhydrases.............................................................................. 23

2.6.1 General properties........................................................................ 232.6.2 CA VI........................................................................................... 26

3 Aims of the present study 314 Materials and methods 33

4.1 Saliva, milk and tissue samples (I–IV).................................................. 334.2 Purification of carbonic anhydrase isoenzyme VI (II) .......................... 344.3 Production and characterisation of rabbit anti-rat CA VI serum (II)..... 344.4 Immunoblotting (I–IV) .......................................................................... 354.5 Immunohistochemical staining (I–IV)................................................... 354.6 Histochemical staining of CA activity (I).............................................. 364.7 Polypeptide sequencing and molecular mass analysis (III)................... 374.8 Deglycosylation of purified CA VI (III)................................................ 374.9 Immunofluorometric assay (III) ............................................................ 374.10 Characterisation of CA VI dehydration activity.................................... 37

5 Results 395.1 Binding of CA VI to enamel pellicle..................................................... 395.2 Dehydration kinetics of CA VI.............................................................. 395.3 Expression and secretion of CA VI by human and rat von Ebner’s

glands..................................................................................................... 405.4 Secretion of CA VI into milk ................................................................ 415.5 Expression and secretion of CA VI in the lower airways...................... 42

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6 Discussion 436.1 Delivery of CA VI into the gastrointestinal tract and its

physiological role there.......................................................................... 436.2 Delivery of CA VI into the respiratory tract and its physiological role

there ....................................................................................................... 457 Conclusions 478 General comments and future considerations 49

References 51

Original publications 63

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1 IntroductionCarbonic anhydrases (CAs) catalyse the reversible hydration of carbon dioxide:H2O + CO2 ↔ H+ + HCO3

-. This reaction is the basis of the most important acid-base buffer system in mammals (Pocock & Richards 2001). The thirteen activeCAs known today have overlapping functions, which, at least to some degree,explains the fact that only a few exact functions of single CA isoenzymes havebeen identified. Carbonic anhydrase isoenzyme VI (CA VI) is the only secretoryCA (for review see e.g. Pastorekova et al. 2004). Its functions have beenconnected to dental caries, acid injury of the gastrointestinal mucosa, and taste andsmell dysfunction in clinical studies (Parkkila et al. 1997, Henkin et al. 1999a, b,Kivelä et al. 1999). Patients with low salivary CA VI concentrations aresusceptible to dental caries and acid injury of the gastroesophageal mucosa, aswell as a decreased sense of taste and smell. The first two pathogenicconsequences are hypothesised to result from decreased CA activity, whereas thedefects in sensory functions are probably attributable to different mechanisms ofaction. An intracellular form of CA VI (CA VIB) has been found in the cytosol,but its enzymatic activity and function have not been studied (Sok et al. 1999).

Since CA VI is the only secreted CA, it is probably expressed in severalglands and their secretory products. In previous studies, CA VI has been found inthe saliva, salivary glands, lacrimal glands, pancreas, nasal glands, serum, bovinemilk, oesophagus, hair follicle, forestomach, large intestine, enamel organ, mucosaof the upper alimentary tract and liver (Fernley et al. 1979, Parkkila et al. 1990,Ogawa et al. 1995, 2002, Kivelä et al. 1997b, Fujikawa-Adachi et al. 1999,Ichihara et al. 2003, Murakami et al. 2003, Ishimatsu-Tsuji et al. 2005, Kubota etal. 2005, Kaseda et al. 2006, Kasuya et al. 2007, Nishita et al. 2007). This thesisprovides novel findings on the expression and enzyme kinetics of CA VI as well asfunctional linkages suggesting that CA VI is a widely expressed constituent ofexcretory fluids and a potent catalyst for pH regulation.

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2 Review of the literature

2.1 Saliva

Human saliva is secreted into the oral cavity from the salivary glands, primarily bythe paired large salivary glands (parotid, submandibular and sublingual glands).Numerous minor salivary glands are distributed all over the oral submucosa. Mostof them are comprised of only one acinus but four larger groups can also beidentified. In the posterior tongue beneath the taste buds lie the von Ebner’s glandsand further back lie the posterior lingual mucous glands. Paired accessory parotidglands are occasionally situated in the cheeks along the parotid ducts. The salivaryglands consist of tubuloacinar secretory endpieces and a branching ductal system.Acinar and tubular endpieces are comprised of serous and mucous cells,respectively. Serous cells produce protein-rich saliva while mucous cells producecarbohydrate-rich saliva. Mixed endpieces contain serous demilune cells amongthe mucous cells. The ductal cells modify the ion concentrations of saliva byabsorbing water and ions and secreting ions (Burkitt et al. 1993). The secretionrate and composition of saliva follow circadian periodicity (Parkkila et al. 1995,Kivelä et al. 1997b) and are controlled by the autonomic nervous system. Tastestimuli affect the saliva composition and secretion rate (Froehlich et al. 1987). Thetotal daily output of saliva is approximately 0.5–1.5 litres. Non-stimulated salivahas a mean flow rate of 0.3 ml/min whereas mastication or tastant stimulatedsaliva is secreted at the rate of about 2 ml/min (Kivelä et al. 1999, Humphrey &Williamson 2001). The flow rate and composition of saliva vary among people.Low flow rates are associated with dental caries and a sensation of oral dryness(Leone & Oppenheim 2001, Pedersen et al. 2005). Hyposalivation denotes ameasurably lowered flow rate of saliva whereas xerostomia denotes a subjectivefeeling of oral dryness (Nederfors 2000).

Whole saliva is composed of secretory products of salivary glands, oral bacte-ria, their metabolic products, blood components diffused through crevices and oralepithelial cells. About 99% of whole saliva is water. Although saliva is initiallyisotonic to serum, the ductal cells absorb sodium and chloride. This renders wholesaliva hypotonic compared to serum but rich in potassium, chloride, sodium, bicar-bonate, phosphate and magnesium. The mean protein concentration of wholesaliva is 1.15 g/l and the pH is 7.3 (Streckfus et al. 1994, Kivelä et al. 1999). Sali-vary α-amylase and proline-rich proteins constitute most of the protein in saliva

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whereas histatins, statherin, lysozyme, lactoferrin, peroxidases, secretory immuno-globulin A and CA VI are present in lower concentrations.

Saliva has several functions in the gastrointestinal tract. The importance ofsaliva to oral well-being is demonstrated in patients with hyposalivation who haverampant caries, a feeling of oral dryness, and difficulty in swallowing and spea-king (Christensen et al. 2001, Pedersen et al. 2005, for review see Soto-Rojas &Kraus 2002). Saliva coats the mucosal surfaces, lubricating and protecting them(Bradway et al. 1989, 1992, Pedersen et al. 2002). It also contains growth factorsthat are essential for the oral mucosa and rinses food debris and bacteria from toothand mucosal surfaces (Lipps 2000, Morris-Wiman et al. 2000, Markopoulos et al.2001, Gröschl et al. 2005). Several factors of saliva (e.g. histatins and peroxidases)possess antimicrobial activity (De Smet & Conteras 2005, Ihalin et al. 2006). Sali-vary minerals are important in maintaining tooth integrity. Acidity derived fromfoods, drinks and bacterial metabolism is buffered by saliva. Saliva also preventsacid injury to the gastrointestinal tract mucosa (Helm et al. 1984). The mostimportant buffering system in saliva is based on bicarbonate, although other signi-ficant systems are based on phosphate and protein (Helm et al. 1982, Bardow et al.2000a, b). A low salivary buffering capacity is associated with high DMFT indexscore (Kivelä et al. 1999).

Specific salivary proteins attach to the dental enamel forming a 0.3–1.1 µmthick protein layer termed the acquired enamel pellicle (Kousvelari et al. 1980,Al-Hashimi & Levine 1989, Amaechi et al. 1999). In vitro studies have shown thatthe pellicle prevents demineralisation of the enamel caused by cariogenic bacteriaand acidity (Zahradnik et al. 1976, 1977, 1978, Featherstone et al. 1993, Nekras-hevych & Stösser 2003). The pellicle matures over time and 7-day-pellicle decrea-ses demineralisation more effectively than 1-day-pellicle (Sönju & Rölla 1973,Zahradnik et al. 1979, Featherstone et al. 1993, Skjorland et al. 1995). However,incubation of teeth with saliva for only three minutes forms a pellicle that decrea-ses demineralisation as effectively as a 2-hour pellicle (Hannig et al. 2004). Themechanism by which the pellicle protects teeth from demineralisation appears toresult from the neutralisation of acid that restricts hydrogen diffusion to the enamel(Hannig & Balz 2001, Nekrashevych & Stösser 2003).

2.2 Dental caries

Dental caries is tooth decay caused by oral bacteria. Oral bacteria build up dentalplaque on the pellicle that together comprise a dental biofilm. The biofilm matures

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over time enabling e.g. wide bacterial diversity, pH gradient and formation offunctional matrix (Garcia-Godoy & Hicks 2008). Some strains of bacteria inbiofilm, especially Streptococcus Mutans, produce substantial amounts of acid asan end product of their sugar metabolism (Kleinberg 2002). The solubility constantof hydroxyapatite, the principal mineral of enamel, is increased by acidity (Larsen& Jensen 1989). Saliva, enamel and pellicle neutralise biofilm borne acids to someextent but considerable and long-lasting acid exposure causes demineralised carieslesions (Zahradnik et al. 1976, 1977, 1978, Nekrashevych & Stösser 2003).Accordingly, patients with high caries prevalence have lower initial dental biofilmpH and lower minimum dental plaque pH after sugar consumption plus slowerrecovery time to initial pH after sugar consumption than caries-free patients(Stephan 1944). However, the association of dental plaque pH with cariesprevalence has not been found in some other studies (Fejerskov et al. 1992, Donget al. 1999). Minor caries lesions, especially the ones not extending to the dentin,may remineralise if favourable surroundings are restored (Zahradnik 1979). Onsome instances even large lesions may remineralise. But if the lesion extendsthrough the enamel and oral bacteria infect the dentin the patient’s need forrestorative dental care is probable (Fejerskov & Kidd 2003).

Dental caries is a common global health problem (Petersen 2003). In Finland37% of adolescents reported tooth ache during the preceding two years (Honkalaet al. 2001). Most of the dental care is related to caries. In 2005 the dental careexpenditure was 606 million euros comprising 5.1% of our total health-careexpenditure and 0.4% of gross domestic product in Finland (Matveinen & Knape2007). A subgroup of 20–25% of children has 50% of all caries (Winter 1990).The high caries prevalence of these particular individuals results from several fac-tors e.g. diet, oral microbiota, personal oral hygiene plus saliva composition andflow rate.

2.3 Taste perception

Taste stimuli dissolved in saliva are converted into afferent neural signals byneuroepithelial taste receptor cells. The stimuli elevate intracellular calcium levelsand trigger the release of neurotransmitters to the proximity of gustatory afferentnerve fibres. These transmit the signal to the central nervous system where theactual taste perception takes place (for review see Lindemann 2001, Gilbertson &Boughter 2003). Taste receptor cells are located in taste buds which are distributedall over the oral mucosa but most of them are found in the tongue (Avery 1987).

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About one fourth of all taste buds are situated in the tops of fungiform papillae thatare located all around the oral cavity. Nearly all of the remaining three quarters oftaste buds are situated in the lateral walls of foliate and circumvallate papillae. Ineach taste bud there are 50 to 150 individual cells of which 10 to 14 are tastereceptor cells. They don’t divide but new cells arise from the proliferative basalcell layer (Stone et al. 1995). The space where taste receptor cell microvilliprotrude to the oral cavity is called a taste pore. The microvilli are rich inchemoreceptors and ion channels that react to taste stimuli. Taste sensation isconcentration-dependent but also the size of the taste stimulus application areaaffects sensation (Smith 1971). Five basic tastes can be perceived: sour, salty,bitter, sweet and umami. Single taste receptor cells can respond to several tastestimuli (Gilbertson et al. 2001). Responses to sweet, bitter and umami tastes aremediated by receptor proteins (Bernhardt et al. 1996, Lin & Kinnamon 1999, Zhaoet al. 2003, Pronin et al. 2004). Salty taste reception is mediated by directmovement of the cations sodium, potassium, ammonium and calcium to tastereceptor cells (Kloub et al. 1997). Sour taste reception is a result of acidification ofa subset of taste receptor cells, although acid-sensing ion channels have beenproposed to take part in sour sensing as well (Miyamoto et al. 1998, 2000, Lyall etal. 2001, Stevens et al. 2001, for review see DeSimone et al. 2001). Weak organicacids (e.g. acetic acid and dissolved carbon dioxide) are believed to enter tastereceptor cells more readily than protons, because they elicit stronger reception andperception of sourness plus lower intracellular pH in taste receptor cells than theirvirtual acidity would suggest (Lyall et al. 2001, Lugaz et al. 2005).

Systemic carbonic anhydrase inhibition causes alterations in basic taste sensa-tions when carbon dioxide containing beverages are ingested (Graber & Kelleher1988, Miller & Miller 1990). In rats the inhibition of carbonic anhydrase causesinhibition of carbon dioxide sensing (Komai & Bryant 1993, Simons et al. 1999,Lyall et al. 2001, 2002). Rat taste buds contain CA activity and CA II has beenlocalised there (Brown et al. 1984, Daikoku et al. 1999). Interestingly, CA activityis also present in the lingual von Ebner’s glands that excrete saliva to the bottom ofthe trenches surrounding the taste receptor cell-rich circumvallate and foliatepapillae (Brown et al. 1984). The function of von Ebner’s glands is closely asso-ciated with taste reception, above all in dissolving and diluting the stimuli andwashing the clefts (Gurkan & Bradley 1988, Sbarbati et al. 1999). Removal ofsubmandibular and sublingual salivary glands damages all taste receptor cells inthe dorsal tongue but not those located in the circumvallate papillae suggesting that

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the saliva of the von Ebner's glands is an important source of growth factors fortaste receptor cells (Morris-Wiman et al. 2000).

2.4 Milk

The mammary glands are paired modified sweat glands located within thesubcutaneous tissue. The development of the mammary glands is completedduring pregnancy as the glandular tissue increases and the amount of fat andconnective tissue decreases. After parturition the alveoli become dilated with milkand have only a thin epithelial lining. The secretory process for milk proteins ismerocrine and for lipids apocrine. The constituents vary during pregnancy andsubsequent nursing. At the end of the pregnancy and for 1–5 dayspostgestationally the mammary glands secrete protein- and lactose-rich colostrum.Thereafter the protein content of the milk decreases, and it is called mature milkthat contains 88% water, 7% carbohydrate, 3.5% lipids, 1.5% protein and ions(particularly sodium, potassium, chloride, calcium and phosphate). Milk proteinsare classified into three major groups: caseins, whey proteins and mucins (fatglobule membrane proteins). The mucin concentration of milk remains the sameduring lactation but the whey-to-casein ratio varies from 9:1 in early lactation to1:1 in late lactation. The total protein concentration of milk gradually decreasesfrom 15 g/l in early lactation to 8 g/l at 6 months and onwards (Lönnerdal 1976,Kunz & Lönnerdal 1992). Colostrum has especially high levels of immunologicalcomponents. Milk not only provides nutrients to the infant but also possessesgrowth factors and components that protect the infant from viral, yeast andbacterial infections (Dewey et al. 1995, Shi & Kong 2000, Lee et al. 2004).β-lactoglobulin, α-lactalbumin and caseins α1-2, β and κ constitute most of themilk protein. The caseins take part in curd formation which lengthens the time themilk is in the stomach. β-lactalbumin induces lactose synthesis but the function ofβ-lactoglobulin is uncertain (for review see Threadgill & Womack 1990,Kontopidis et al. 2004). Several other proteins are found in minor amounts in milk.Some of them are growth promoting like epidermal growth factor that stimulatesthe proliferation of intestinal cells (Chang & Chao 2002, Takeda et al. 2004).Others, for example lactoferrin, have an anti-microbial effect (van der Strate et al.2001).

Some intact milk proteins can be found in the stools of breastfed infants sug-gesting that they have enzymatic, immunological or other functional significancethroughout the gastrointestinal tract of the infant (Davidson & Lönnerdal 1987).

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Interestingly CA activity has been found in histochemical studies of the goat mam-mary gland and the amount of activity correlates with the volume of secreted milk(Cvek et al. 1998).

2.5 Structure, function and development of the respiratory tract

The lower airways consist of the trachea, the primary bronchi and the lungs. Theluminal surfaces of airways are covered by mucosa and surrounded by thesubmucosa. The mucosal epithelium consists of ciliated cells, mucous goblet cells,brush cells, small granule cells and basal cells. In rats there are also serous cells inthe epithelium. In humans, serous cells are found in the tracheal epithelium only inthe foetus. The submucosa is composed of a relatively loose connective tissue thatalso contains seromucous glands. Their ducts penetrate the mucosa. The surfaceliquid on the airway mucosa is produced predominantly by the submucosal glands,but also by the epithelial secretory cells. The airway surface liquid can be dividedinto the periciliary liquid layer and the mucus covering it. Inhaled small particles,e.g. bacteria, attach to the mucus and are deployed to the larynx by a coordinatedsweeping motion of the cilia. Cilia are flexible membrane extensions of the cellthat are found in vast numbers on the respiratory tract luminal surfaces (more than10 million per square millimetre). The low viscosity of the periciliary liquid isessential for the function of cilia (for review see Boucher 2004). The airwaysurface liquid also possesses antibacterial proteins and properties (Nakayama et al.2002, for review see Verkman et al. 2003).

The trachea divides gradually into several smaller airways (bronchi, bronchio-les, respiratory bronchioles, alveolar ducts and alveoli). Simultaneously themucosal epithelium becomes cuboidal and may be ciliated. The height of the epit-helial cells and the number of seromucous glands decreases as the lumen becomessmaller in diameter. There are also some goblet cells, brush cells, dense-core gra-nule cells and protein-secreting Clara cells. The airways end in gas exchangingunits called alveoli. They are composed of type I and II pneumocytes with somemacrophages and brush cells. The type II pneumocytes are secreting cells produ-cing the surfactants that e.g. prevent the adhesion of alveoli walls during expira-tion. The walls of the alveoli are very thin in parts. Between the inhaled air and theblood there are only three layers including an extremely thin type I pneumocyte,the basal lamina of the alveolar epithelium and the capillary endothelium (Burkittet al. 1993).

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2.6 Carbonic anhydrases

2.6.1 General properties

Carbonic anhydrases are proteins that catalyse the reversible hydration of carbondioxide: H2O + CO2 ↔ H+ + HCO3

- (for review see Lindskog 1997, Pastorekova etal. 2004). CAs have various functions e.g. in pH homeostasis, CO2 transfer and ionexchange (for review see Chegwidden et al. 2000). Three gene families named α,β and γ have been recognized. In vertebrates the α-CAs are dominant(Hewett-Emmett 2000). So far thirteen active α-CAs and three genetically related,but acatalytic CAs, have been identified in vertebrates. Isoenzymes CA I-III, CAVIB, CA VII and XIII are cytosolic, CA V A and B are mitochondrial, CA VIA issecreted and CA IV, CA IX, CA XII, CA XIV and CA XV are membrane-bound(Fig. 1) (Pastorekova et al. 2004, Hilvo et al. 2005). The nuclear protein nonO/p54nrb has CA activity but only little sequence homology to CAs and thereforenonO/p54nrb is considered a non-classical CA (Karhumaa et al. 2000).

Fig. 1. Location of the active α-CA isoenzymes in a vertebrate cell.

The genes of the membrane-bound and secreted α-CAs form a group that hasevolved separately from the cytosolic and mitochondrial α-CAs (Fig 2) (Jiang &Gupta 1999, Hewett-Emmett 2000).

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Fig. 2. Phylogenic table of α-CAs.

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The catalytic site is conserved in the active CAs (Hewett-Emmett & Tashian1996). Three conserved histidine residues (His94, 96, 119) bind a zinc ion (Berg et al.2002). The zinc ion binds water lowering the ionisation constant pKa of water from15.7 to 7. In the first reaction step of CA catalysed carbon dioxide hydration, thezinc-bound water ionises by releasing one of its protons to form a CA boundOH--group as in [1]: (Chegwidden & Carter 2000)

[1]: CA-Zn2+-H2O CA- Zn2+-OH- + H+

In the second reaction step [2] the CA bound OH--group attacks CO2 to produceHCO3

-, which is then replaced by water as in [3]:

[2]: CA- Zn2+-OH- + CO2 CA- Zn2+- HCO3-

[3]: CA- Zn2+-HCO3- + H2O CA- Zn2+-H2O + HCO3

-

The properties of the buffer solution affect the catalytic activity of CAs byaccelerating the diffusion or release of protons (Berg et al. 2002). Some buffers aretoo large to reach the active site of CAs. CAs possess proton shuttle structures thatenhance the movement of protons between the active site of the enzyme and theouter surface (Tu et al. 1989, Duda et al. 2001, Fisher et al. 2005). The buffer andthe proton shuttle enable CA II to catalyse the hydration of carbon dioxide ahundred times faster than in their absence (Berg et al. 2002). The hydration ofcarbon dioxide occurs spontaneously at a rate of kcat=0.15 s-1 (kcat/Km=0.0027 M-1

s-1), but CAs accelerate the reaction to over a million molecules per second(Khalifah 1971, Berg et al. 2002). The uncatalysed rate of bicarbonate dehydrationkcat is 50 s-1 but CAs are physiologically significant because they catalyse thedehydration rate to an even faster velocity (Tsuruoka & Schwartz 1998, Berg et al.2002). The enzyme activity profile for the hydration of carbon dioxide as afunction of pH has been characterised for CA isoenzymes I–VA, VII, IX and XII(Khalifah 1971, Heck et al. 1994, Baird et al. 1997, Hurt et al. 1997, Earnhardt etal. 1998, Ulmasov et al. 2000, Wingo et al. 2001, Nishimori et al. 2005). The pHprofile is a saturation curve that maintains near-maximal values in the pH range7.5–9 whereas the values decrease considerably as a function of acidity below pH7.5. CA II has very high activity (kcat=1.4 x 106 s-1 and kcat/Km=1.5 x 108 M-1 s-1)whereas CA III has low activity. The other enzymes have 20–70% of the activityof CA II (Wingo et al. 2001, Nishimori et al. 2005, Hilvo et al. 2008). In pH 5.5–

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7.5 the kinetic constants kcat/Km and kcat for the CA catalysed dehydration ofbicarbonate increase as a function of acidity. The maximal value for kcat/Km of CAIV for dehydration is 2.8 x 107 M-1 s-1 in pH 5.5, being 2–3 times higher than thevalue for CA II and CA VII (Baird et al. 1997, Earnhardt et al. 1998).

2.6.2 CA VI

Two subtypes of CA VI are known. The A-type is thus far the only known secretedmember of the α-CA family and the B-type a stress-induced cytoplasmic form(Sok et al. 1999, Kubota et al. 2005). They have separate promoters but thepolypeptides differ only in that the B-type lacks the signal peptide (Sok et al.1999). The B-type has been found from mouse embryonic fibroblasts, salivaryglands and ameloblast-derived cell lines (Sok et al. 1999, Kubota et al. 2005). Sofar there is no information about CA activity in the B-type, and therefore in thisthesis, the A-type is called simply CA VI. CA VI has also been called gustin,although Thatcher et al. (1998) reported that gustin and CA VI are in fact the sameprotein.

The gene encoding CA VI is located in the tip of the short arm of chromosome1 (Sutherland et al. 1989). Human CA VI has 68%, 71.5% and 74% amino acidsequence identity with sheep, ovine and canine CA VI, respectively (Aldred et al.1991, Jiang et al. 1996). In the C-terminus of CA VI there is a 10 amino acidextension compared to cytosolic CAs (Aldred et al. 1991). Human CA VI has 308amino acids that include a 17 amino acid leader sequence in the N-terminus(Aldred et al. 1991). Bovine, sheep and canine CA VI have about 10 amino acidsmore in the C-terminus than does the human CA VI (Fernley et al. 1988b, Aldredet al. 1991, Jiang et al. 1996). According to the cDNA sequence of human CA VIthe molecular size of the mature polypeptide backbone is 33.6 kDa (Aldred et al.1991). Using the predicted polypeptide sequence, the mature bovine CA VI poly-peptide has been estimated to be 35.5 kDa (Fernley et al. 1988b, Jiang et al. 1996).Using SDS-PAGE the size of the mature CA VI has been estimated to be 37–46kDa (Feldstein & Silverman 1984, Kadoya et al. 1987, Murakami & Sly 1987,Fernley et al. 1988a, Parkkila et al. 1990, Ogawa et al. 1992, 2002, Kivelä et al.1997b, Thatcher et al. 1998, Kimoto et al. 2004). CA VI has two potential N-gly-colysation sites and oligosaccharide side chains that total 4–9 kDa (Murakami &Sly 1987, Fernley et al. 1988a, Thatcher et al. 1998). Different glycosyltransfera-ses present in the parotid and submandibular glands produce different glycoformsof CA VI (Hooper et al. 1995a, b). CA VI appears also as a 7–12 fold larger mole-

26

cule, suggesting that the enzyme forms homologic complexes (Fernley et al. 1979,1988a). There is a disulphide bond between cysteine residues Cys25 and Cys207 ofovine CA VI (Fernley et al. 1988b).

The catalytic activity for hydration by CA VI has been reported to be mode-rate with kcat/Km=4.9 x 107 M-1 s-1 (Feldstein & Silverman 1984, Fernley et al.1988a, Nishimori et al. 2007a, b). CA VI has been found from saliva, salivaryglands, lacrimal glands, pancreas, nasal glands, serum, bovine milk, oesophagus,hair follicles, forestomach, large intestine, enamel organ the mucosa of upper ali-mentary tract and liver (Table 1) (Fernley et al. 1979, Parkkila et al. 1990, Ogawaet al. 1995, 2002, Kivelä et al. 1997b, Fujikawa-Adachi et al. 1999, Ichihara et al.2003, Murakami et al. 2003, Ishimatsu-Tsuji et al. 2005, Kimoto et al. 2004,Kubota et al. 2005, Kaseda et al. 2006, Kasuya et al. 2007, Nishita et al. 2007).CA VI is primarily expressed in the serous acinar cells but is also expressed in theductal cells (Parkkila et al. 1990, Ogawa et al. 1993, Ichihara et al. 2007).

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Table 1. Expression of CA VI in mammalian tissues.

The concentration of CA VI in saliva varies diurnally and has also a highindividual variation (Kivelä et al. 1997a, b). During sleep the CA VI concentrationis very low in saliva and serum, while it increases after awakening followed bybreakfast (Kivelä et al. 1997b). The mean CA VI concentration in whole saliva is1.8 mg/L (0.1–10.0 mg/L) for non-stimulated and 5.0 mg/L (0.6–16mg/L) forstimulated whole saliva. Most of the CA VI in saliva is secreted by the parotidgland (Fernley et al. 1991). Interestingly, low salivary CA VI concentrations have

Tissue Species Immuno-histochemistry

Westernblot

mRNA Immunoassay

Parotid gland Humanrat, caninesheep

+ + RT-PCR

6.4 mg/g

Submandibular gland

Humanratcaninemousesheepbovine

+ + RT-PCR

1.8 mg/g0.29 mg/g

Minor salivary glands

canine RT-PCR

Saliva Humansheep, ratbovine

+ 5.0 mg/l7.8 mg/l

Mammary gland bovine + RT-PCR 0.40 µg/g

Milk bovine 7.9 mg/l

Nasal glands mouse + + RT-PCR

Lacrimal glands sheep, rat + + RT-PCR 4.2 µg/g

Serum Humanbovine

+ 0.20 mg/l2.1 mg/l

Oesophagus caninebovine

+ RT-PCR

Enamel organ rat RT-PCR

Pancreas Human RT-PCR

Large intestine bovine + (+)

Liver bovine RT-PCR 0.92 µg/g

Hair follicle mouse cDNA micro-array hybridisation, In situ hybridisation

Upper alimentary tract mucosa

canine + RT-PCR

Zygomatic gland canine + RT-PCR

Sublingual gland canine + RT-PCR

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been shown to be associated with an increased risk of acid injury to thegastroesophageal mucosa and high DMFT index values (Parkkila et al. 1997,Kivelä et al. 1999), although low CA VI concentrations are not associated withlow saliva pH (Kivelä et al. 1999). Low salivary CA VI concentrations are alsoassociated with a decrease in taste perception and an increase in apoptosis of tastereceptor cells in the circumvallate papilla taste buds (Henkin et al. 1999a, b).Therefore CA VI has also been suggested to act as a growth factor. However, nodirect evidence is available to support this suggestion.

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3 Aims of the present studyCarbonic anhydrase isoenzyme VI is the only known secreted CA isoenzyme.Earlier findings have demonstrated that the enzyme is present in saliva and tearfluid where it is proposed to participate in maintaining the acid-base homeostasisof the gastrointestinal tract lumen and surface of the eye, respectively. Thehypotheses of this thesis are that CA VI is a general constituent of exocrine glandsecretions and a factor protecting teeth and the mucosae of the alimentary andrespiratory tracts. The specific aims were:

1. To determine the distribution of CA VI in rat and human exocrine glands.

2. To study whether CA VI is bound to the human enamel pellicle and to assessits possible physiological role there.

3. To determine the bicarbonate dehydration activity of human CA VI.

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4 Materials and methods

4.1 Saliva, milk and tissue samples (I–IV)

Collection of the saliva and tissue samples was carried out according to theprovisions of the Declaration of Helsinki and informed consent was obtained fromeach volunteer. Human whole saliva was collected from healthy volunteers intotest tubes. Saliva from human von Ebner’s glands was collected from two healthyvolunteers’ circumvallate papillae with a micropipette and from foliate papillaewith nitrocellulose sheets (Spielman et al. 1993). Frozen human milk was obtainedfrom the Oulu University Hospital Milk Centre. Colostrum samples were obtainedfrom 9 mothers on the 2–4th day postpartum. Samples from saliva drops wereobtained at the same time period from two 3-day-old infants prior to their breastfeeding. Mature milk samples were also obtained from four mothers on the 90th

day postpartum. Human tongue and parotid gland specimens were obtained fromthe Department of Forensic Medicine, University of Oulu and lingual carcinomasamples of patients undergoing surgery at the Clinic of Otorhinolaryngology, OuluUniversity Hospital.

All animal experiments described below had the approval of the Animal Careand Use Committee of the University of Oulu. Rat saliva was collected under ana-esthesia and pilocarpine-stimulation. Rat mammary gland specimens were takenfrom sexually mature non-pregnant female rats and from rats two days before orafter parturition. Rat trachea and lung samples were collected from foetuses andfrom 1-, 2-, 5-, 10-, 20-, 30-day-old and adult male rats. Tongue and salivary glandsamples were collected from adult male rats.

The tissue samples used for protein purifications and immunoblotting werehomogenised in ice-cold 0.1 M Tris-SO4 buffer pH 8.7, containing either 1 mMphenylmethanesulphonylfluoride, 1 mM benzamidine and 1 mM o-phenanthrolineor protease inhibitors from Complete™ (Roche Applied Science) which wereapplied according to the manufacturer’s instructions. The samples were then cen-trifuged and the supernatants were used for the experiments. Samples for immuno-histochemistry were fixed in Carnoy’s fluid or 4% paraformaldehyde in PBS for18 h at 4 °C, and embedded in paraffin, and sections of 5 µm were subjected toimmunohistohemical stainings.

Permanent non-carious human teeth extracted in routine dental care wereobtained from the Department of Oral and Maxillofacial Surgery, University ofOulu and from the Parolannummi Garrison Hospital. Three methods were used to

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produce enamel pellicle. 1) Prior to extraction the teeth were polished with pumiceand pellicle was allowed to form in situ for 2 h. 2) Crown halves were polishedwith pumice and incubated in vitro in saliva or in PBS in a rotary shaker for 2 h atroom temperature. 3) Crown quarters were polished with pumice and incubated ina rotary shaker for 2 h at room temperature with purified human CA VI (3 or 9 mg/mL in PBS) or with purified salivary α-amylase (230kU/L in PBS) or in PBSalone. Enamel pellicle proteins were collected for immunoblotting experimentsfrom two volunteers using a curette.

4.2 Purification of carbonic anhydrase isoenzyme VI (II)

An affinity gel matrix was prepared by coupling the CA inhibitor p-aminomethylbenzenesulphonamide to CM Bio-Gel A (Bio-Rad) according to Khalifah et al.(1977). Samples were diluted with buffer containing 0.1 M Tris-SO4 and 0.2 Msodium sulphate at pH 8.7 and incubated with inhibitor affinity gel matrix inconstant rotation. After the incubation the gel was washed first with buffer solutioncontaining 0.1 M Tris-SO4, 0.2 M sodium sulphate, 1 mM benzamidine, and 20%(v/v) glycerol, pH 8.7 followed by the same solution at pH 7.0. CA VI was elutedfrom the inhibitor affinity gel matrix with a specific elution buffer containing 0.4M NaN3, 0.1 M Tris-SO4, 1 mM benzamidine, and 20% (v/v) glycerol, pH 7.0. Theeluted proteins were dissolved in SDS loading buffer and then subjected to SDS-PAGE followed by Coomassie Blue staining.

4.3 Production and characterisation of rabbit anti-rat CA VI serum (II)

The purified rat salivary CA VI was dissolved in sodium dodecyl sulphate samplebuffer and the proteins were separated using SDS-PAGE (Laemmli 1970). Themajor 41 kDa band representing the mature glycolysed CA VI was used for theimmunisation of rabbits. Anti-rat CA VI serum was tested using immunoblots andimmunoprecipitation. The antiserum identified two major polypeptides of 41 and37 kDa in rat saliva and purified rat salivary CA VI preparations (II). Themolecular masses of these bands correspond to the masses of the glycosylated andpartially glycosylated forms of CA VI reported earlier for human CA VI(Murakami & Sly 1987). A minor 56 kDa polypeptide was found in rat saliva (II).Preimmune serum did not recognize any polypeptide bands in the controlimmunoblots. To further analyse the antiserum, the antibodies were coupled to

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Protein A Sepharose CL-4B (Pharmacia Biotech), which was subsequentlyincubated with a saliva sample. The bound proteins were eluted using SDS loadingbuffer and then subjected to SDS-PAGE. All three abovementioned bands werefound in the eluate obtained from the Protein A Sepharose CL-4B containing CAVI antibodies (data not shown). To obtain CA VI antiserum free from antibodies tothe 56 kDa proteins, 500 µg of rat saliva was transferred onto a PVDF sheet andthe 56 kDa band detected by immunoblotting was cut out. Then 2.5 µl rabbit anti-rat CA VI serum diluted in 500 µl 1% BSA-PBS was incubated with the PVDFband stripe bearing the 56 kDa proteins for 24 h in constant shaking at roomtemperature. The resulting supernatant (later 56 kDa absorbed antiserum to rat CAVI) identified only the 41 and 37 kDa protein bands in rat saliva (data not shown).The anti-human CA VI serum and anti-rat CA II serum have been characterisedearlier (Parkkila et al. 1990, Kaunisto et al. 1995). Antibody to human salivary α-amylase was purchased from Sigma Chemicals.

4.4 Immunoblotting (I–IV)

The proteins were separated on SDS-PAGE and thereafter electroblotted ontonitrocellulose or PVDF sheets according to a standard method (Towbin et al.1979). The sheets were washed first with Tween-20 containing buffer, incubatedthen with blocking solution (Tween-20 containing buffer with BSA or colostrum)followed by incubating with primary antiserum. The sheets were subsequentlywashed with Tween-20 containing buffer, treated with blocking solution, washedbriefly with Tween-20 containing buffer and incubated with peroxidase-conjugated anti-rabbit immunoglobulin G. After extensive washing with Tween-20containing buffer the bands were visualised with enhanced chemiluminescence(ECL) protein visualising method according to the manufacturer’s instructions.

4.5 Immunohistochemical staining (I–IV)

The 5µm-thick sections for immunoperoxidase staining were deparaffinised andpre-treated with 3% (v/v) H2O2. After incubation with blocking solution (1% (w/v)BSA-PBS or cow colostrum whey or 1% (v/v) goat colostrum) the sections wererinsed with PBS, incubated with antiserum diluted in 1% (w/v) BSA-PBS, rinsedagain with PBS and re-incubated with biotin-conjugated swine anti-rabbitimmunoglobulin G diluted in 1% (w/v) BSA-PBS. Subsequent to rinses with PBSthe sections were incubated with streptavidin-conjugated peroxidase diluted 1:600

35

in PBS and rinsed with PBS. Finally, the enzyme localisation was visualised byincubating the sections with a solution containing 9 mg of 3,3’diaminobenzidinetetrahydrochloride in 15 ml of PBS + 5 µl of 30 % (v/v) H2O2 and rinsing withPBS. The sections for immunofluorescence staining were deparaffinised and pre-treated with 1% (w/v) BSA-PBS. After rinsing with PBS, the sections wereincubated with antiserum diluted in 1% (w/v) BSA-PBS, rinsed with PBS, re-incubated thereafter with rhodamine-conjugated swine anti-rabbitimmunoglobulin G diluted in PBS and rinsed with PBS. All sections wereexamined with a Nikon Eclipse E600 microscope and photographed using a NikonCoolpix 950 digital Camera. The images were further processed with Corel Drawand Adobe Photoshop computer software.

4.6 Histochemical staining of CA activity (I)

A pellicle was formed on tooth samples by incubating them in saliva or withpurified salivary CA VI. Control samples were incubated in PBS alone. Thesamples were thereafter stained for CA activity using the Hansson’s method(Hansson 1967) briefly as follows:

1. Incubation of the samples for 1 min with washing buffer solution containing 9mL 1/15 M KH2PO4 and 1 mL 1/15 M Na2HPO4 in 1 L of saline.

2. Rinsing of the samples for 8 min with fresh Hansson’s medium (solutioncontaining 1 mL 0.2 M CoSO4, 6 mL 0.5 M H2SO4 and 10 mL 1/15 MKH2PO4 added to a freshly prepared solution of 0.75 mg NaHCO3 in 40 mLdistilled water). The medium was changed continuously to enable air contactof the samples. Carbon dioxide was blown over the surface of the CoSO4-containing solution for 10 min prior to addition of the NaHCO3-containingsolution and over the samples during the rinsing.

3. Washing the samples for 1 min with the washing buffer containing 9 mL 1/15M KH2PO4 and 1 mL 1/15 M Na2HPO4 in 1 L of saline.

4. Incubation of the samples for 5 s with a freshly prepared solution containing1% (NH4)2S in distilled water.

5. Incubation for 1 min with the washing buffer containing 9 mL 1/15 MKH2PO4 and 1 mL 1/15 M Na2HPO4 in 1 L of saline.

36

Another set of control samples were incubated in Hansson’s medium containingthe carbonic anhydrase inhibitor acetazolamide at a final concentration of 50 mM.

4.7 Polypeptide sequencing and molecular mass analysis (III)

The peptide sequence analyses were performed using matrix-assisted laserdesorption ionisation mass spectrometry followed by analysis with the ProFoundand PeptideSearch programs. The molecular mass of purified CA VI from humanmilk was analysed using Voyager DE RP matrix-assisted laser desorptionionisation time-of-flight mass spectrometer analysis (PerSeptive Biosystems).

4.8 Deglycosylation of purified CA VI (III)

CA VI samples purified from human milk and saliva were deglycosylated with N-glycosidase F (Roche diagnostics) as described earlier (Petäjä-Repo et al. 1991)and analysed on SDS-PAGE.

4.9 Immunofluorometric assay (III)

Concentrations of CA VI in colostrum (n=9), mature milk (n=4) and 3-day-oldinfants’ saliva samples (n=2) were measured using competitive time-resolvedimmunofluorometric assay as described earlier (Parkkila et al. 1993). The meanintra-assay coefficient of variation of the present series was 9.4% and theinterassay coefficient of variation determined in three assays was 9.7%.

4.10 Characterisation of CA VI dehydration activity

The initial velocities of bicarbonate dehydration were determined by stopped-flowspectrophotometry measuring the change in absorbance of a pH indicator at 25 °C(Khalifah 1971). The final concentrations of bicarbonate varied from 1.3 to 50mM. The buffer-indicator pairs used were p-nitrophenol (pKa 7.10) / 3-(N-morpholino) propanesulphonic acid (pKa 7.02) for pH 7 and chlorophenol red (pKa

6.03) / 2-(N-morpholino) ethanesulphonic acid (pKa 6.05) for pH 6.5, 6 and 5.5.During the measurements the ionic strength was maintained at 0.2 M by theaddition of Na2SO4 to the solution. The mean initial rate was determined from 8–10 reaction traces comprising the initial 10% of the reaction. The measurements inpH 6.5 were repeated twice using two different concentrations of CA VI. The

37

uncatalysed rates were subtracted, and the kinetic constants kcat, Km and kcat/Km

were determined by a nonlinear least-squares method (Enzfitter, Biosoft).

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5 Results

5.1 Binding of CA VI to enamel pellicle

In immunohistochemical staining, CA VI specific immunoreactivity was found inboth the in vivo and in vitro formed enamel pellicle (I). The supragingival enamelsurface showed a uniform peroxidase reaction (I). The staining intensity of in vitroformed enamel pellicle was dependent on CA VI concentration, suggesting aconcentration-dependent enamel binding profile for CA VI (I). Interestingly, theCA VI bound to the enamel was found to display carbonic anhydrase activity inhistochemical staining (I). Human enamel pellicle proteins subjected toimmunoblotting with CA VI antiserum revealed a 38.5 kDa polypeptide band,while normal rabbit serum revealed no positive staining (I).

5.2 Dehydration kinetics of CA VI

The pH dependence of the kinetic constants kcat, kcat/Km and Km are shown in figu-res 3–5. The maximal values for dehydration by CA VI were 3.0 x 105 s-1 for kcat

and 2.9 x 107 M-1s-1 for kcat/Km (Figs 3 and 4). The Km for dehydration ranged from5.2–16 mM (Fig. 5) (unpublished results). The dehydration kinetics of CA VI aresimilar to those of its closest genetic relatives, the membrane-bound CA isoen-zymes IV, XII and XIV (Baird et al. 1997, Earnhardt et al. 1998).

Fig. 3. pH dependence of kcat for dehydration of bicarbonate.

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Fig. 4. pH dependence of kcat/Km for dehydration of bicarbonate.

Fig. 5. pH dependence of Km for dehydration of bicarbonate.

5.3 Expression and secretion of CA VI by human and rat von Ebner’s glands

Immunochemical staining demonstrated that the serous acinar cells of both thehuman and rat von Ebner’s salivary glands in the posterior tongue expressed CAVI. In rat, the serous demilune cells of the posterior lingual mucous gland andductal cells of both glands also expressed CA VI. The ductal content of both rat

40

glands stained strongly for CA VI, suggesting that both glands also secrete theenzyme into saliva (II). Immunostaining for CA VI was also seen in the rat tastepores and in unidentified cells within the taste buds (II). The staining pattern in ratlingual salivary glands was identical when the 56 kDa absorbed antiserum to ratCA VI was used (results not shown). Immunoblotting of the von Ebner’s glandextract and saliva confirmed the presence of CA VI in the von Ebner’s glands andthe secretion into saliva. The antiserum to human CA VI recognized a 40 kDapolypeptide in immunoblots of von Ebner’s gland saliva collected from theorifices of circumvallate and foliate papillae (II). An additional weaker 56 kDapolypeptide band was recognized in saliva collected from the foliate papilla orifice(II). The antiserum to rat CA VI recognized 41 kDa and 37 kDa polypeptide bandsin immunoblotting of the CA preparation purified from rat von Ebner's glands (II).Sections and immunoblots stained with normal rabbit serum or preimmune serumshowed no staining, confirming the specificity of CA VI immunostaining (II).

5.4 Secretion of CA VI into milk

The alveolar epithelium of rat mammary glands were found to express CA VI, andthe enzyme was also present in the postpartum alveolar milk (III). CAs purifiedfrom human milk showed one major 42 kDa polypeptide band in SDS-PAGE thatwas recognized by antiserum to human CA VI (III). The molecular mass of themajor band corresponds to the glycosylated form of salivary CA VI. In massspectrometry, the accurate size of the glycopeptide was 37.8 kDa. Occasionally aminor band of 29 kDa was purified from milk corresponding to CA II which likelyoriginates from red blood cells contaminating the milk. The molecular weights ofglycosylated and deglycosylated CA VI purified from human milk and saliva wereidentical (III). Rat mammary glands showed specific bands at 56 kDa, 42 kDa and36 kDa in immunoblots (III). The most intense bands were found in the lactatinggland, a moderate band in glandular tissue prior to parturition, and a faint band inthe resting gland (III). The analysis of the protein sequence data of CA VI purifiedfrom human milk revealed 100% identity with human salivary CA VI (40%coverage) (III). The mean concentration of CA VI in colostrum was 34.7 mg/l(range 10.0–78.4 mg/l, n=9) and in mature milk 4.5 mg/l (2.6–6.9 mg/l, n=4) (III).The CA VI levels in the saliva of two infants were 1.9 and 3.6 mg/l (III).

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5.5 Expression and secretion of CA VI in the lower airways

The serous cells of submucosal tracheobronchial glands, the ductal cells of theglands, the serous cells of tracheobronchial mucosal epithelium and bronchiolarClara cells were found to express CA VI (IV). CA VI was also present in theductal content of the glands. Interestingly, CA VI was localised in the base of thecilia in the bronchiolar ciliated cells (IV). The expression of CA VI in theglandular cells appeared as soon as the glandular structures were detected, in the20th postnatal day. In contrast, the Clara cells expressed CA VI from the 1st

postnatal day onwards. A similar staining pattern was obtained when the 56 kDaabsorbed antiserum to rat CA VI was used (results not shown). Sections stainedwith anti-rat CA VI serum absorbed with purified rat CA VI or preimmune serumshowed no positive staining, confirming the specificity of the immunostaining(IV).

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6 Discussion

6.1 Delivery of CA VI into the gastrointestinal tract and its physiological role there

CA activity in human saliva was reported for the first time over 60 years ago byRapp (1946). This activity was demonstrated to result from CA VI secreted by thelarge salivary glands (Fernley et al. 1979). The results here show that CA VI isdelivered into the gastrointestinal tract not only in saliva secreted by the largesalivary glands but also in von Ebner’s glands’ saliva and in milk. Saliva and milkcontain inorganic compounds and several proteins that affect overall homeostasisin the oral cavity and locally on tooth surfaces. Patients with a low salivary flowrate, like patients suffering from hyposalivation, have usually rampant carieswhich points to the fact that saliva protects teeth from caries. The importance ofsalivary CA activity for dental health was first suggested by Szabo (1974) whoreported that low CA activity in saliva is associated with high caries prevalenceand later by Kivelä et al. (1999) who reported that low concentrations of CA VI insaliva are associated with high DMFT index scores. Salivary buffering capacityand pH are two long-known caries-associated factors. Interestingly, Kivelä et al.(1999) found, however, no correlation between salivary CA VI concentrations andsalivary buffering capacity or pH. The finding here showing that CA VI attaches tothe enamel in a concentration-dependent manner and as an active form suggeststhat CA VI functions rather as a local caries protective factor in the enamel pellicleby neutralising the acidity produced by the bacteria in the dental biofilm. In fact,Kimoto et al. (2006) recently showed that inhibiting CA activity in a mixture ofsaliva and oral bacteria yields lower pH after sugar exposure than in non-CA-inhibited control mixtures. The solubility constant of enamel hydroxyapatiteincreases as pH decreases and hence long-lasting acidity on enamel dissolves itresulting in caries lesions. The acidity on enamel results principally from bacterialsugar metabolism in the dental biofilm. Thus, CA VI located in the enamel pellicleis in an optimal position for neutralisation of the acidity produced by the dentalbiofilm. More importantly, the present results show that the catalytic activity ofCA VI has a high maximal kcat (3.0 x 105 s-1) for dehydration of bicarbonate. Also,CA VI has a low dehydration Km that is similar to the saliva bicarbonateconcentration (Bardow et al. 2000a, b). To conclude, the results here suggest thatCA VI bound to enamel facilitates locally the removal of the dental biofilm

43

produced acidity as carbon dioxide by catalysing the reaction: H+ + HCO3- → H2O

+ CO2.Henkin et al. (1999a, b) have reported that CA VI has an important role in

taste perception. They found that low concentrations of CA VI in saliva areassociated with lowered taste perception and morphological deterioration of thetaste buds of the circumvallate papillae and that oral zinc administration elevatedsalivary CA VI concentration and restored the normal morphology (Henkin et al.1999a, b). A zinc deficient diet is also associated with lowered trigeminal nerveresponses to carbonated water (Komai et al. 2000). The connection of CAs to tasteperception is also demonstrated in studies by Miller and Miller (1990), McMurdoet al. (1990), and Graber and Kelleher (1988) who showed that oral use of CAinhibitors causes a multitude of side-effects on taste perception e.g. decrease insensing carbonated water. Carbon dioxide sensing recovers fast after topicalapplication of the CA inhibitor dorzolamide on the dorsum of the tonguesuggesting that salivary CA VI plays a major role in carbon dioxide sensing but isnot the only CA involved as the taste buds also express other CAs e.g. CA II(Daikoku et al. 1999, Simons et al. 1999). The taste buds are abundantly located inthe walls of the trenches surrounding lingual circumvallate and foliate papillae.Since whole saliva can not readily enter the trenches the source of CA VI in thetrenches is principally, as the present study suggests, the von Ebner’s glands,which are located beneath the papillae and open directly into the bottom of thetrenches. The importance of the von Ebner’s glands to the functions of taste budsof circumvallate papillae is also supported by a report that shows that the removalof the rat submandibular and sublingual glands results in damage to the taste budslocated in the dorsum of the tongue but not in those located in the circumvallatepapillae (Morris-Wiman et al. 2000). In the trenches, CA VI may remove excessprotons or carbon dioxide by catalysing the dehydration/hydration reaction andCA VI may thereby affect taste perception by modifying the actions of the proton-gated sodium and potassium channels or decreasing the concentration of carbondioxide that readily penetrates the plasma membrane of the taste receptor cells.Acid neutralisation may also protect taste receptor cells from apoptosis oralternatively, CA VI may be a trophic factor for the taste bud cells as has beenproposed by Henkin et al. (1999a, b). The trophic effects of CA VI may bemediated via the widely expressed family of calmodulin dependent cyclicnucleotide phosphodiesterases, some of which have been shown to be activated byCA VI (Law et al. 1987, Omori & Kotera 2007).

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Low salivary CA VI concentrations are also associated with acid injury of thegastrointestinal mucosa suggesting that CA VI is involved in pH balancing on themucosa (Parkkila et al. 1997). CA VI retains its activity in the low pH present ingastric juice and hence the acid neutralisation effect of CA VI likely extends fromthe stomach to the intestinal tract as well (Parkkila et al. 1997). For maximal acidneutralisation it would be beneficial if CA VI would be immobilised on theluminal surface of the mucosal epithelia e.g. by receptor proteins as proposed byHooper et al. (1995a, b). The present study shows that CA VI is delivered into thegastrointestinal tract not only in saliva but also in milk and particularly in highconcentrations in colostrum. In the gastrointestinal tract, CA VI possibly hasseveral physiological effects alone or together with other milk proteins. Milkbicarbonate concentration (3.5–8.3 mM) (Anderson 1992) is near the dehydrationKm of CA VI (5.2–16 mM) enabling efficient catalysis by CA VI. The finding thatCA VI appears in particularly high concentrations in colostrum suggests that CAVI originating from milk possibly has other mechanisms of action in addition tothe neutralisation effect in the newborn gastrointestinal tract. Concentrations ofmany whey proteins, especially of immunoglobulins, are also high in colostrum(Korhonen et al. 2000, Ballabio et al. 2007). Since CA VI is known to form acomplex with immunoglobulin G in serum (Kivelä et al. 1997b) it may also formcomplexes with immunoglobulins in milk and thereby participate in the anti-microbial functions of milk in the newborn’s gastrointestinal tract. Milk CA VImay also have similar trophic effects in the gastrointestinal tract as has beenproposed for salivary CA VI on the taste receptor cells (Henkin et al. 1999). Thusit is apparent that CA VI can act as a multifunctional protein like e.g. lactoferrin inthe gastrointestinal tract (Ward et al. 2005) and have a specific role not only intooth and mucosal protection and taste perception but also in the development ofthe newborn gastrointestinal tract, and that the role of milk CA VI is tocompensate for the low CA VI levels in the newborn’s own saliva.

6.2 Delivery of CA VI into the respiratory tract and its physiological role there

In the respiratory tract, efficient defence mechanisms of the mucosa and strictlyregulated cell renewal systems are needed to surmount various chemical, physicaland microbial stresses. The present results show that CA VI secreted by the serouscells of the tracheobronchial glands and bronchiolar Clara cells can participate inthese defence and renewal processes of the mucosa. Secreted CA VI is likely

45

linked to acid removal from mucosal surfaces as carbon dioxide by catalysing thereaction: HCO3

- + H+ → CO2 + H2O. A neutral pH on the mucosa is important forthe mucosal defence against microbes in at least two ways. First, acidity reducesbactericidal activity in the surface liquid of human airway epithelia and henceincreases their susceptibility to bacterial infection (Nakayama et al. 2002). Sec-ond, acidity lowers the beat frequency of bronchial cilia (Clary-Meinesz et al.1998), which also increases the susceptibility of the airway epithelia to bacterialinfection (Afzelius 1976, Whitelaw et al. 1981). Accordingly, CA VI could beimplicated in protection of the mucosa by increasing the bactericidal activity of thesurface liquid and/or increasing cilia motility via pH neutralisation of the surfaceliquid. Interestingly, CA VI is localised in the base of the cilia suggesting that itsfunction is locally linked with cilia motility. However, the lack of significant acidpredispositions in the respiratory tract also suggests alternative roles and mecha-nisms of action for CA VI there. For instance, CA VI could have trophic functionsas proposed for CA VI in the gastrointestinal tract. The fact that CA VI is presentin airway surface liquid and in milk points to the fact that CA VI is not only a con-stituent of saliva and tear fluid but also a more general constituent of glandularsecretions.

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7 Conclusions1. CA VI attaches to the acquired enamel pellicle in a concentration-dependent

manner and in an enzymatically active form. In the enamel pellicle, CA VI isin an optimal location to neutralise the acidity produced by the dental biofilmand thus to protect the teeth from caries.

2. CA VI displays a marked bicarbonate dehydration activity and a low Km. Thedehydration activity constant kcat/Km of CA VI is the highest of all CAs. TheKm values are close to the bicarbonate levels found in milk and salivasuggesting that CA VI is well able to accelerate the pH neutralisation in milkand saliva plus on tooth and mucosal surfaces.

3. CA VI is secreted into the mammalian oral cavity not only by the largesalivary glands but also by the serous cells of lingual von Ebner’s salivaryglands and posterior mucous glands. The saliva of the von Ebner’s glands isdelivered into the trenches surrounding the taste bud-rich circumvallate andfoliate papillae suggesting that CA VI is implicated in the taste stimulimodifying effect of the von Ebner’s saliva and might also protect the tastereceptor cells from apoptosis.

4. CA VI is secreted into mammalian milk in high concentrations. Theparticularly high concentrations of CA VI in colostrum suggest that milk CAVI has a specific significance in the physiology and development of thenewborn gastrointestinal tract.

5. CA VI is also secreted into the lower airways by the serous cells of thesubmucous gland. CA VI is also present in tracheobronchial epithelia,bronchiolar Clara cells and in the bases of the cilia of the ciliated epithelialcells of the lower airways. In the lower airways, CA VI may have otherfunctions, in addition to its classical role in the regulation of pH homeostasis,such as trophic or anti-microbial effects or effects on ciliary motility.

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8 General comments and future considerationsThe results presented here provide supporting data on the hypothesis that CA VI isa potent acid neutraliser in the enamel pellicle. The data explains, at least in part,why a group of patients with high salivary CA VI concentration has a highresistivity to caries. Accordingly, oral hygiene products e.g. artificial salivas couldbe supplemented with CA VI. Especially patients who suffer from hyposalivationderived rampant caries could benefit from these products. However, additionaldirect studies are necessary to confirm the cariesprotective effect and the exactmechanism of action of CA VI. CA VI deficient mice, follow up patient studiesand in vitro demineralisation experiments combined with specific CA VI inhibitorsthat do not hinder biofilm metabolism are essential to address the unresolvedquestions.

Also the second hypothesis that CA VI is a general constituent of exocrinegland secretions was proved valid. It was suggested that CA VI functions ongastrointestinal and respiratory tract mucosa principally as an acid neutraliser butalso other mechanisms of action should be considered. As has been suggestedearlier CA VI may be a multifunctional protein with e.g. growth factor properties.The high concentration of CA VI in mammalian milk, especially in colostrum,suggests further that CA VI has a significant role in the development of thenewborn gastrointestinal tract. The CA VI deficient mice are expected to providecomprehensive answers to these questions, although double CA deficient micemay be needed since other CAs may compensate for CA VI deficiency.

49

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Original publications

This thesis is based on the following articles, which are referred to in the text bytheir Roman numerals:

I Leinonen J, Kivelä J, Parkkila S, Parkkila AK & Rajaniemi H (1999) Salivarycarbonic anhydrase isoenzyme VI is located in the human enamel pellicle.Caries Res 33:185–190.

II Leinonen J, Parkkila S, Kaunisto K, Koivunen P & Rajaniemi H (2001)Secretion of carbonic anhydrase isoenzyme VI (CA VI) from human and ratlingual serous von Ebner’s glands. J Histochem Cytochem 49:657–662.

III Karhumaa P, Leinonen J, Parkkila S, Kaunisto K & Rajaniemi H (2001) Theidentification of secreted carbonic anhydrase VI as a constitutive glycoproteinof human and rat milk. Proc Natl Acad Sci USA 98:11604–608.(Karhumaa P and Leinonen J contributed equally to this work)

IV Leinonen J, Saari K, Seppänen J, Myllylä H & Rajaniemi H (2004)Immunohistochemical demonstration of carbonic anhydrase isoenzyme VI(CA VI) expression in rat lower airways and lung. J Histochem Cytochem52:1107–1112.

Additionally, unpublished original data on CA VI are included.

Reprinted with permission from S. Karger AG, Basel (I), The HistochemicalSociety (II, IV), National Academy of Sciences, U.S.A. (copyright 2001) (III).

Original publications are not included in the electronic version of the dissertation.

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