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CURRENT VIEWS OF COLLAGEN DEG RADATION

Progress Towards Understanding the Resorption of Connective Tissues Gillian Murphy and John J. Reynolds

Summary

Collagen is the most abundant vertebrate protein and forms a stable fibrous architecture in connective tissues, such as bone, cartilage, skin and tendon. Much recent research has been directed towards an understanding of the molecular and cellular mechanisms involved in the synthesis and degradation of collagen, because a change in the normal balance, or an increased destruction of collagen, can cause loss of function of specialized tissues. This short retliew attempts to summarize present knowledge about the proteolytic destruction of collagen and the exciting new work on the factors that are emerging as important controlling agents. An understanding of the regula- tory processes is yielding some important clues regarding the levels at which they can faiI in pathological conditions and we speculate about evolving strategies for precen ting uncontrolled resorption.

The Structure and Biochemistry of Collagen

Collagen constitutes approximately 50% of the protein of a fully developed vertebrate organism. It exists primarily as fibres whose function is to provide a chemically and physically stable sup- porting network in the matrix of connective tissues. Collagenous com- ponents are also integral parts of the architecture of basement membranes and collagen molecules have important functions in developmental processes, cell attachment and cell movement.’ In the normal adult organism, collagen turnover is very low (half-life 50-300 days, depending on the tissue) but is significantly accelerated during devel- opment, tissue remodelling (such as involution of the uterus, part-partum) and wound healing. Diseases such as arthritis have long been known to be associated with the breakdown of the extracellular collagenous matrix of cartilage and bone and when collagen

cannot be renewed the condition can be crippling. In some kinds of cancer, collagen degradation must occur in order to permit tumour cell invasion and metastasis within the host.

The collagens consist of a number of closely related proteins, both chemically and functionally. The most common collagen is referred to as t y p I and is the major structural protein of bone, tendon and skin. The collagen of cartilage is called type I1 and another collagen that is more abundant in vascular and embryonic tissues is type 111. Together these three types are referred to as interstitial collagens: they are all composed of triple helical polypeptide chains with each chain being just over 1000 amino acids long; unusually they contain the amino acid hydroxyproline and glycine is placed at every third residue. The most common collagen, type I, has a triple helix composed oftwo slightly different chains (two of one type and one of the other) and they are usually notated as [al(I) 21 a&). In addition to the more abundant interstitial collagens there are now more than a dozen other types of collagens, occurring in much smaller amounts, associated with different types of cell and of less well understood function. These collagen types are often referred to as cell-associated collagens. The individual polypeptide chains (a-chains) making up each different type of collagen are derived from separate genes and exhibit subtle variations in both sequence and post-translational modifi- cations which are reflected in their specific roles and tissue distribution.3

The triple helical structure of collagen is extremely resistant to the action of most proteinases. Varying susceptibility to proteolysis by collagenolytic enzymes results largely from differences in the tertiary and quaternary structure, deter- mined not only by the sequence, but also by the presence of covalently bound carbohydrate and the type of inter- and

intra-molecular cross links, which vary from tissue to tissue and with the stage of mat~rat ion.~ A further factor is the association of collagen with ‘ground substance’ macromolecular materials, such as proteoglycan, elastin and certain other glycoproteins, which may mask the proteolytically sensitive region of collagen molecules.

Proteolytic Destruction of Collagen

In in vitro experiments collagens can be degraded by enzymes from all four classes of proteinases (aspartic, cysteine, metallo- and serine active sites).5. Their relative roles in vivo seem to vary from situation to situation depending on the tissue environment, type of cell and physiological state and whether inflam- matory mediators are present. In most cases the evidence for the precise mechanisms of collagen turnover is still equivocal and controversial. Indeed there are still some workers who are as yet unconvinced that proteolytic en- zymes are important! Current views are that the initial step in degradation is an extracellular proteolytic process and involves a loosening up of the highly cross-linked insoluble collagen fibres of the matrix. The involvement of mechan- ical disruption and free radicals remains uncertain but might be contributory processes.

Connective tissue cells have the ability to synthesize and secrete a group ofmetalloproteinases (MPs) which func- tion at neutral pH and can digest the major macromolecules of connective tissue mat rice^.^^^ This group of protein- asesis therefore the most likely candidate for an important role in matrix turnover. The group includes a specific collagenase which cleaves the three types of intersti- tial collagens (Fig. I , route a), as well as at least two other MPs with broader substrate specificities, being capable of degrading other connective tissue com-

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COLLAGEN FIE=

By gelatinase and

E XTRACELLULAR ENDOCYTOSIS OF FIBRILS EXTRACELLULAR

After enzymatic or By specific collagenase mechanical damage By cysteine and

serine proteinases \

\ DIGESTION

By cathepsins B, L, N at acid pM in

phagolysosomes

--

I FRAGMENTATION r Bv various other proteinases proteinases

Fig. 1. Possible pathways for the intra- and extracellular digesiion of collagenfibrils. It is considered likely that most degrada tion is initiated by proteolytic attack on collagen fibrils. Extracellular degradation may proceed either by way of tissue metalloproteinases (route a ) or by serine and cysteine proteinases from specialized injlammaiory cells such as polymorphonuclear leucocytes (route c). At various stages modi$ed collagen (and other extracellular matrix macromolecules) may be taken up by phagocytic cells (route b) and further degraded intracellularly within phagolysosomes.

ponents but not interstitial collagens. One of these MPs is proteoglycana~e~

Action of Specific Collagenase

(also called stromeljkn) which can Specific collagenase has long been degrade basement membrane collagen considered to be the key enzyme in as well as proteoglycans and another, ~ollagenolysis.~~~ It is a Zn2+ metallo- gelatinase, can degrade denatured colla- endopeptidase cleaving the native triple gens. Proteinases with similar specificity helix of the interstitial collagens at a are produced in small amounts by single locus in each constituent a-chain; macrophages and are secreted by the the active form from most mammalian polymorphonuclear leucocytes, neutro- tissues has an M , of about 45,000. phils and eosinophils. Such enzymes Figure 3 illustrates the action of colla- would generate collagen fragments genase on tropocollagen (type I colla- which could be phagocytosed by connec- gen) : tropocollagen is relatively tive tissue cells and degraded within the immature collagen and is used for most phago-lysosomal system (Fig. 1, route biochemical experiments because it can b). Indeed, there are many demonstra- be extracted from young tissues by tions of fragments of collagen fibrils within cells at sites of rapid resorption (Fig. 2). Weakening of the collagen fibrillar structure by mechanical proces- ses of wear and tear, and by the action of serine and cysteine proteinases de- rived from polymorphonuclear leuco- cytes (which act as ‘cross-linkases’, see below) would also permit phagocytosis of fibrils.

The relative importance of the extra- and intra-cellular routes cannot be stated with any certainty. The serine proteinases (as well as collagenase) from polymorphonuclear leucocytes and rela- ted inflammatory cells constitute a powerful threat to collagen molecules and they may be considered a separate extracellular pathway (Fig. 1, route c). In some instances, it is also possible that lysosomal cysteine proteinases may be secreted by cells. Although theseenzymes function at more acid pH values, in certain situations a limited acidic peri- cellular environment may exist which would permit local degradative

acetic acid and can be reconstituted as pseudo-fibrils with essentially no inter- molecular cross-links. Type I collagen is cleaved across all three chains at a Gly-Ile bond in the q(1) chains and a Gly-Leu in the a,(I) chain, these being located in a region of low helix stability, of the way along the molecule from the

NH, terminus. That collagenase cleaves only one bond in each chain and has only a very limited ability to cleave Gly-Leu and Gly-Ileu in other proteins suggests that the precise orientation of this bond within the stable collagen helix is essential for efficient enzyme activity.

The form of collagenase produced by different connective tissue cells and macrophages is similar, if not identical, but that from polymorphonuclear leuco- cytes (packaged in the bone marrow myelocytes) appears to be slightly different in some of its biochemical properties, and is probably a separate gene product. Although collagenases are efficient in the degradation of the individual tropocollagen molecules into 4 and 4 fragments, they cannot disrupt the covalent intermolecular cross-links which are present in intact fibrils. In test-tube experiments with collagen the pure collagenase appears, therefore, to have a much reduced ability to break down collagen fibrils. It is only within the context of the in uiuo situation that its full potential can be realised when it acts in concert with other proteinases. We have attempted to illustrate the attack of collagenase on cross-linked fibrils in Fig. 4 (pathway a). When mas-

activity.6 Fig. 2 Intracellular collagen fibrils in a pig synovial cell involved in matrix resorption. (Photograph courtesy of Dr. Audrey Glanert.)

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1 Collagenase

1 Denaturation at 37 “C

Fig. 3. The action of specijic collagenase on type I tropocollagen. Tropocollagen is prepared by the extraction of young tissues with acetic acid and after pur$cation it can be reconstituted as fibrils with very few cross-links between chains. This ‘monomer’ unit consists of three a-chains in a helix and in type I collagen, the most common collagen, there are two slightly dyerent chains that are referred to as a, (-, two chains) and as (--, one chain). The diagram illurtrates a single cross-link between an a, and an a, chain. Collagenase cleaves across all three chains to give characteristic t (TCA) and f (TCB) pieces. The products produced by collagenase vary depending upon where the actual cross-links are located, but the essential feature is that the fragments denature at physiological temperatures.

sive resorption takes place the matrix becomes almost denuded of collagen fibrils (Fig. 5; contrast this with the normal tissue illustrated on the front cover). The native state of the collagen molecule relies heavily on the associa- tion with other molecules and degraded portions of the helix quickly lose their triple helical conformation. They are then susceptible to the action of a num- ber of less specific proteinases including MPs from connective tissue cells, par- ticularly gelatinase, as well as serine proteinases from polymorphonuclear leucocytes and lysosomal proteinases. Gelatinase from both connective tissue cells and polymorphonuclear leucocytes has been shown to accelerate collagen- ase activity on reconstituted collagen fibrils and insoluble collagen prepara- tions.

It has been suggested that tumours may synthesise special MPs that degrade basement membranes,’” particularly basement membrane collagen type IV, and that their production may be correlated with metastatic potential. Whether such activities are different from the MPs synthesised by normal cells is presently unclear, but they are undoubtedly related proteinases and the considerations that we give below to collagenase would still be relevant.

Possible Role of Cysteine and Serine Proteinases

Also illustrated in Fig. 4 (pathway 6) is the action on fibrils of the battery of potent serine proteinases secreted by polyrnorphonuclear leucocytes. Particu- larly important is elastase, which has the ability to degrade the non-helical regions at the ends of tropocollagen molecules

where the inter-molecular cross links are sited (‘cross-linkase’). Although their action in vivo would be limited to situations where inflammatory cells were present (which includes a number of examples of rapid collagen turnover, such as the early phases of tissue injury and uterine involution) these proteinases could ostensibly generate phagocytos- able free a-chains.

The lysosomal cysteine proteinases, cathepsins B, L and N (and the aspartic proteinase, cathepsin Db, which only function at rather acid pH, also may act on the cross-link regions of collagen, possibly within a specially generated environment outside the cell, e.g. the ruffled border of osteoclasts and other membrane/fibre interfaces. There is some evidence that they can also degrade the uncross-linked native helix at acid pH values.6

Regulation of Collagen B tea kdown

An overview of the possible regulatory mechanisms in collagen breakdown is shown in Fig. 6. One of the most striking facts regarding the MPs of both connec- tive tissue cells and macrophages is that they are ‘inducible’, i.e. their synthesis and secretion seem to be controlled by factors modifying the cell’s behaviour. Some transformed cells may synthesize MPs without further stimulation. Ex- periments carried out in vitro have contributed greatly to our knowledge

b a b a

Denaturation and further attack by proteinases such as gelatinase

1 Denaturation and further cleavage

within modified a-chains

1

Fig.4. Action of collagenase and other proteinases on collagen fibrils. This diagram attempts to illustrate the action of collagenase (pathway a) and serine and cysteine proteinases (pathway b) on collagen fibrils that have intra- and inter-molecular cross-links as they occur in vivo. The arrangement of tropocollagen molecules and subsequent inter-molecular cross-linking is three-dimensionaP but the diagram can only be a two-dhensional representation. Pathway a illustrates the kin& of product that might be generated by collagenase and which would then become susceptible to other proteinases. Pathway b shows the elimination of cross-links (‘ cross-linkase’ activity) before further attack takes place on denatured a-chains.

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Fig. 5. Extracellular matrix with only a few intact collagenfibrils in tissue (pig synovium) undergoing rapid resorption.

and have shown that collagenase pro- duction can be switched on in cells which normally synthesize connective tissue matrix proteins, including colla- gen, in the resting state. Indeed, under certain conditions, collagenase can become a major gene product.'l Unlike most other proteinases, MPs cannot usually be extracted in any significant amounts from tissues but are synthesized de novo upon stimulation.

Perhaps most relevant to the in uivo situation are the findings that specific hormones appear to play an important role in specific tissues, e.g. parathyroid hormone can induce collagenase produc- tion by osteoblasts but not most other cells. Cell-derived factors have recently been found to be powerful stimuli for MP synthesis by connective tissue cells. Many such activities have been de- scribed and referred to under a variety of names, such as mononuclear cell factor.12 Cytokines are synthesized by lectin or lymphokine-stimulated macro- phages and monocytes, although other cell types including fibroblasts could be important sources. It now seems likely, however, that many of these activities are forms of interleukin 1 (IL 1):13 at concentrations down to lo-" M, it

(Photograph courtesy of Dr. Audrey Glanert.)

stimulates collagenase production by a range of connective tissue cells including chondrocytes and synovial fibroblasts. Other properties include the abilities to induce bone and cartilage degradation as well as the proliferation of T cell populations. For chondrocytes it seems that IL 1 is an important mediator of cell-cell interactions and is a likely mediator of the degradative events in joint damage. Several types of tumour cell and epithelial cells have also been shown to produce cytokines capable of stimulating collagenase production; it is possible that many of these as yet uncharacterized materials will turn out to be closely related to IL 1. Mast cell products such as heparin and histamine may also have the ability to induce collagenase secretion in some cells. In some instances, processes such as cell transformation can stimulate MP production.

Whether the various molecules that induce MPs act at a transcriptional or translational level is not yet known, but the recent development of cDNAs for collagenase14 and other MPs will permit the investigation of the mechanisms for control of enzyme production. Our own work has shown that corticosteroids are

amongst the most potent hormonal inhibitors of MP synthesis and we have proposed that this may be one of the important physiological roles of hydro- cortisone. Specific hormones may also be involved in controlling MPs in other tissues, e.g. progesterone in the uterus, and this area should be fruitful for new research.

The MPs, including collagenase, are secreted from cells in culture in a latent form.l6 Due to the difficulty of obtaining large amounts of purified proteinases, studies of the activation process have been limited. Although proteinases such as trypsin and plasmin can activate the latent MPs, causing a 10-20 K fall in M,., the same effect can be reproduced by incubation with several organomer- curial reagents. It has not been unequi- vocally demonstrated that any peptide material is lost during this activation process, in a way analogous to the acti- vation of proenzymes of the digestive system and of prohormones to hor- mones. The possibility that physiological activators exist has been extensively investigated. One putative activator has been described which tends to co-purify with collagenase,16 but the former also appears itself to be a latent MP, requiring activation. The possibility that the activation mechanism is an autolytic process triggered either by a small molecule such as ESAF" (endo- thelial cell stimulating activity factor) or by membrane or membrane-bound enzyme interaction has also been sug- gested. To date evidence suggests that

CULLAGENASE

inhibitor

ACTIVATION

ACTIVE TO GIVE INACTIVE COLLAGENASE

COMPLEX

BINDING TO I-- SUBSTRATE

Fig. 6. Controlling steps in connective tissue resorption by metalloproteinases. Metalloproteina- ses usually are only synthesized when cells are stimulated by factors such as interleukin 1 (IL 1) and speciJc hormones (control points in nucleus a, and cytoplasm b), whereas the inhibitor, TIMP, seems to be synthesized constitutively by most cells. The relative amounts of TIMP and active enzyme (control points c, d ) determine whether or not resorption takes place. The mechanism of activation of latent enzymes remains an enigma (point d ) but , it seems likely that plasmin vrom plasminogen) may be an important activator.

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the most likely physiological mechanism involves the generation of plasmin by cell-derived plasminogen activators acting on plasminogen.18

Collagenase Inhibitors

Some of the most interesting findings in recent years have been the discoveries of potent specific natural inhibitors of MPS.'~-~* Studies have shown that inhibitors from various tissue sources including cell and tissue culture media, plasma, amniotic fluid, vitreous humour and cartilage extracts are all closely related. We named this type of inhibitor TIMP (tissue inhibitor of metallopro- teinases) and first purified it from human amniotic fluid:23 it is a glycopro- tein of M , 28,000 and combines essen- tially irreversibly with active MPs. Apart from TIMP, the serum proteinase inhibitor a,-macroglobulin is the only other known collagenase inhibitor. Due to its large M , (780 K), the latter is probably confined to sites close to blood vessels, although it may be synthesized by some connective tissue cells.

It has been observed that most cells, especially connective tissue cells, synthe- size TIMP constitutively and we pro- posed that this is a fail-safe me~hanism'~ to 'mop-up' the low levels of MPs which may be produced by resting cells (Fig. 4). Little is known about the control of the levels of TIMP, although production is elevated in cells stimulated to produce MPs by IL 1 or phorbol esters, and by treatment with corticosteroids. We have been able to localise TIMP in cells stimulated by phorbol esters using immunocytochemical methods.e4 Also we have found that cells can simulta- neously produce both TIMP and latent MPs and hence we have concluded that the activation of latent MPs and the subsequent interactions of active en- zymes with either substrates or TIMP must all be extracellular events. MPs will only be active at sites where they are secreted and activated in greater amounts than TIMP, because active species are rapidly sequestered by the inhibitor.15 Enzyme-inhibitor com- plexes cannot be reactivated to any significant extent but the fate of enzyme- TIMP complexes has not yet been deter- mined; they may be phagocytosed like protease nexin-thrombin complexes.

Much of our biological work is now centred on the hypothesis that tissue destruction means an imbalance of active MPs over TIMP and in several model systems we have evidence to suggest the validity of this hypothesis. As yet we do not know precisely the mechanisms that control TIMP produc-

tion but in pathological situations TIMP levels may simply be overcome by the presence of proteinases from inflam- matory cells.

Future Trends

The new information that is now available concerning the mechanisms involved in connective tissue destruction suggest that several avenues of research might be profitably explored.

Firstly, further experiments should be aimed at firmly establishing the impor- tance of cytokines such as IL 1 as mediators of the induction of MPs by cells, as well as their mode of action and the role of second messengers. Analysis of the regulatory mechanisms of cyto- kine production and action and the role of natural inhibitors is required.

Secondly, to obtain a fuller under- standing of the control of expression of MPs and TIMP at all levels from their genes to their extracellular activity, the development of compounds that could alter the balance between the synthesis of TIMP and MPs (such as cortico- steroid derivatives) in a localized environment would be particularly important.

Thirdly, experiments are required to test whether TIMP, or fragments derived from it or synthetic peptides, could be used to control tissue destruction in in viuo model systems. Such experiments may also help to elucidate the precise contribution of MPs to resorption in each particular situation.

Fourthly, further investigations should be made of the mechanisms that lead to the recruitment of polymorpho- nuclear leucocytes in situations where there may be synergism between MPs and other proteinases. The efficacy of specific inhibitors in model systems should help to delineate the role of serine and cysteine proteinases in connective tissue turnover.

We suggest that further research in these areas could lead to new therapies that might be useful not only in arthritis and related diseases but also in control- ling tumour invasiveness.

The work of the authors is supported by funds from the Medical Research Council and the Arthritis and Rheuma- tism Research Council. The authors are indebted to Jelena Gavrilovic and Joan Heath for suggestions about the manu- script and to Christopher C. Green for expertly preparing the diagrams.

R EFER EN CES

1 BORNSTEIN, P. & SAGE, H. (1980). Struc- turally distinct collagen types. Annu. Rev. Biochem. 49,957-1003. 2 MILLER, E. J. (1984). Chemistry of the collagens and their distribution. In Extracel- Iular Matrix Biochemistry (ed. K. A. Piez & A. H. Reddi), pp. 41-81. Elsevier, New York. 3 KIVIRIKKO, K. I. & MYLLYLA, R. (1984). Biosynthesis of thecollagens. In Extracellular Matrix Biochemisiry (ed. K. A. Piez & A. H. Reddi), pp. 83-118. Elsevier, New York. 4 WOOLLEY, D. E. (1984). Mammalian col- lagenases. In Extracellular Matrix Biochemi- stry (ed. K. A. Piez & A. H. Reddi), pp. 119-1 57. Elsevier, New York. 5 BARRETT, A. J. & MCDONALD, J. K. (1980). Mammalian Proteases: A Glossary and Bibliography, vol. 1, Endopeptidases. Academic Press, London. 6 SELLERS, A. & MURPHY, G. (1981). Collagenolytic enzymes and their naturally occurring inhibitors. In International Review of Connective Tissue Research, vol. 9 (ed. D. A. Hall & D. S. Jackson), pp. 151-190. Academic Press, London. 7 SELLERS, A., REYNOLDS, J. J. & MEIKLE, M. C. (1978). Neutral metalloproteinases of rabbit bone. Separation in latent forms of distinct enzymes that when activated degrade collagen, gelatin and proteoglycans. Biochem. J. 171, '493496. 8 MURPHY, G., CAWSTON, T. E., GALLO- WAY, W. A., BARNES, M. J., BUNNING, R. A. D., MERCER, E., REYNOLDS, J. J. & BURGEON, R. E. (198 1 b). Metalloproteina- ses from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin. Biochem. J. 199, 807-81 1. 9 GALLOWAY, W. A., MURPHY, G., SANDY, J. D., GAVRILOVIC, J., CAWSTON, T. E. & REYNOLDS, J. J. (1983). Purification and characterization ofa rabbit bone metallopro- teinase that degrades proteoglycan and other connective-tissue components. Biochem. J.

10 LIOTTA, L. A., TRYGGVASON, K., GARBISA, S., HART, I., FOLTZ, C. M. & SHAFIE, S. (1980). Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Naiure 284,

11 AGGELER, J., FRISCH, S. M. & WERB, Z. (1984). Collagenase is a major gene product of induced rabbit synovial fibroblasts. J. Cell Biol. 98, 16561661. 12 DAYER, J.-M., BREARD, J., CHESS, L. & KRANE, S. M. (1979). Participation of mono- cyte-macrophages and lymphocytes in the production of a factor that stimulates collagenase and prostaglandin release by rheumatoid synovial cells. J. Clin. Invest. 64, 1386-1 392. 13 SAKLATVALA, J., PILSWORTH, L. M. C., SARSFIELD, S. J., GAVRILOVIC, J. & HEATH, J. K. (1984). Pig catabolin is a form of interleukin 1. Biochem. J. 224,461-466. 14 GROSS,R. H.,SHELDON,L. A.,FLETCHER, C. F. & BRINCKERHOFF, C. E. (1984). Isolation of a collagenase cDNA clone and

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15 NAGASE, H., JACKSON, R. C., BRINCKER- HOW, C. E., VATER, C. A. & HARRIS, E. D. (1981). A precursor form of latent collagen- ase produced in a cell-free system with mRNA from rabbit synovial cells. J. Biol. Chem. 256, 11951-1 1954. 16 VATER, C. A., NAGASE, H. & HARRIS, E. D. (1983). Purification of an endogenous activator of procollagenase from rabbit synovial fibroblast culture medium. J. Biol. Chem. 258,93749382. 17 WEISS, J. B., HILL, C. R., MCLAUGHLIN, B. & ELSTOW, S. (1983). Potentiating effect of heparin in the activation of procollagenase by a low-Mr angiogenesis factor. FEBS Lett.

18 WERB, Z., MAINARDI, C. L., VATER, C. A. & HARRIS, E. D. (1977). Endogenous activation of latent collagenase by rheuma- toid synovial cells. Evidence for a role of

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19 REYNOLDS, J. J., MURPHY, G., SELLERS, A. & CARTWRIGHT, E. (1977). A new factor that may control collagen resorption. Lancet ii, 333-335. 20 MURPHY, G. & SELLERS, A. (1 980). The extracellular regulation of collagenase acti- vity. In Collagenase in Normal and Pathologi- cal Connective Tissues (ed. D. E. Woolley & J. M. Evanson), pp. 65-81. John Wiley, London. 21 WELGUS, H. G. & STRICKLIN, G. P. (1983). Human skin fibroblast collagenase inhibitor. Comparative studies in human connective tissues, serum, and amniotic fluid. J. Biol. Chern. 258, 12259-12264. 22 BUNNING, R.A.D., MURPHY, G., KUMAR, S., PHILLIPS, P. & REYNOLDS, J. J. (1984). Metalloproteinase inhibitors from bovine cartilage and body fluids. Eur. J. Biochem. 139, 75-80. 23 MURPHY, G., CAWSTON, T. E. & REY- NOLIX, J. J. (1981~). An inhibitor of colla-

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Radioi mmunoassay and Phylogeny Jerold M. Lowenstein

genase from human amniotic fluid. Purification, characterization and action on metalloproteinases. Biochem. J. 195,

24 HEMBRY, R. M., MURPHY, G. & REY- NOLDS, J. J. (1984). Imrnunolocalization of tissue inhibitor of metalloproteinases (TIMP) in human cells. Characterization and use of a specific antiserum. J. Cell Sci. (In the press.) 25 CAWSTON, T. E., MURPHY, G., MERCER, E., GALLOWAY, W. A., HAZLEMAN, B. L. & REYNOLDS, J. J. (1983). The interaction of purified rabbit bone collagenase with puri- fied rabbit bone mztalloproteinase inhibitor. Biochem. J. 211, 313-318.

167-170.

J. J. REYNOLDS is head of, and G. M U R P H Y a member of. the Cell Physiology Department, Strangeways Research Laboratory, Cambridge CB1 4RN, VK.

M olecu I a r

Summary

Traditionally, phylogenetic relations among living and extinct species have been estimated f rom their morphology, particularly that of the bones and teeth. During the past lwo decades, molecular comparisons of D N A , RNA and proteins have increasingly influenced the tax- onomy of living forms. Recently, radio- immunoassay (RIA) has been applied to the resolution of phylogenetic disputes b y testing the relationships of residual fossil proteins with those of living organisms.

Introduction

Traditional taxonomy has been plagued by the problem of convergent evolution : anatomical similarities between two or mcre groups of organisms not closely related genetically. A classic example is the resemblance of various Australian marsupials to specific placental mam- mals. To the European immigrants it seemed that there were marsupial wolves, cats, mice, pigs, and flying squirrels, though the common ancestor of placentals and marsupials lived more than a hundred million years ago. Conversely, morphology can diverge relatively rapidly when new environ- mental opportunities open up. Compare, for instance, the morphological con- servatism of turtles and crocodilians

during a two hundred million-year period with the differentiation of mam- mals into such diverse creatures as elephants, moles, bats, whales and monkeys during the past seventy million years.

It is not surprising that problems of convergence and differing rates of morphological change have produced voluminous and essentially unresolvable conflicts among taxonomists. Similar anatomy does not necessarily establish genetic closeness, nor does dissimilar anatomy necessarily prove great remoteness.

This frustrating reality has given rise, among other phenomena, to the taxo- nomic approach known as cladism,’ which consists of carefully measuring and quantitating anatomical features and sorting them into a hierarchy of traits labelled ‘primitive’ or ‘derived’. In this manner, it is hoped to avoid the pitfalls of convergence and parallelism. In some quarters, cladistics has been elevated nearly to religious status as the answer to all taxonomic dilemmas but, as we shall see, 40 dental measurements may be no better than one for distin- guishing convergence from shared de- scent.

Molecular Evolution

All the genetic information that deter- mines growth, development and mor- phology is contained in the genome. DNA produces RNA which encodes proteins. Therefore, in seeking genetic similarities, why not compare the DNA or RNA or proteins of different species rather than their bones and teeth? During the past twenty or so years, molecular comparisons of this sort have played an increasing role in the tax- onomy of organisms from bacteria to man.2-4 In most cases the results have confirmed classical morphometric rela- tions, but inevitably there have been surprises, such as the demonstration (through comparisons of their RNA) that the ar~haebacteria~ are a third form of life, distinct from eubacteria and eukaryotes, and that man is so closely related to the African apes, the chimp- anzee and gorilla (as first determined by serum protein immunology), that they probably diverged from a common African ancestor about 5 million years ago? much more recently than most anthropologists would have believed possible .

A variety of molecular techniques have been applied to the taxonomic in- vestigation of living organisms: protein electrophoresis and immunology, DNA sequencing and hybridization, RNA