The Making of Mice and Men A comprehensive review of the scientific literature

surrounding the Zonadhesin protein

An introduction to the structure, isoforms, evolution, function, binding and

localization of the protein most widely thought to control species specific

fertilization

2015

Alicia Wafa Hewlett-Packard

1/1/2015

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Literature Review of Zonadhesin Research

INTRODUCTION

This literature review aims to summarize the relevant research concerning the protein

Zonadhesin, as well as to provide an overview of the knowledge of the protein by subject in

chronological order. Research over various aspects of this protein are explained herein,

including: the domain structure, the isoforms identified, factors concerning the evolution of the

protein, the function of the protein, the binding, and the localization of Zonadhesin.

DOMAIN STRUCTURE

This section will summarize the research done between 1995 and 2011 concerning the

domain structure of Zonadhesin. The functions of the individual domains, the order of the

domains, and the biochemical structure of the protein. Because the structure of the protein

alludes to its function, in depth knowledge of the domains and their relationship to each other is

beneficial to a broad understanding of Zonadhesin.

Published in 1995, the paper published by Hardy and Garbers is the first to identify the

Zonadhesin gene. It finds the C terminus end of Zan is 36 amino acids long, very basic, and

intracellular. There is one transmembrane section. The N terminus end of the protein is 2418

amino acids long, and extracellular. There is an N terminus domain, a mucin-like domain, and 5

domains which are analogous to the von Willebrand Factor (D0-D4). D0 is roughly 25% as long

as the other D domains.

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The mucin domain is of particular interest to this paper. Mucin proteins have high

amounts of o-glycosylation, like the protein found in the human intestine, MUC 2, which had

four vWF homologous domains, which helps with cell adhesion. They postulate that the mucin

like domain could help stop the sperm from becoming trapped in the male reproductive tract by

inhibiting cell adhesion, or that it could help promote cell adhesion to the oviducts. The vWF like

domains also seemed to promote oligomerization and may bind to heparin, just as VWF does.

The ZP2-ZP3 proteins believed to bind to Zan have sulfated carbons just like heparin, indicating

this protein may bind here. This paper is part of the foundation for the body of work on this

protein, and is heavily referenced.

In fact, Gao and Garbers (1997) reference Hardy and Garbers (1995) and build from their

research. It investigates the newly discovered Zan protein and searches for it in mice, where the

scientists indeed find Zan. The Mice have the same D domains as the pigs, with a few

exceptions. The mice have a 21 tandem repeat of a portion of D3, whereas the only partial repeat

found in the pigs is of D0. These D domains do appear to be wide spread, appearing not only in

vWF and Zan, but in intestinal mucins of humans, rats and mice as well. Pig Zan had other

tandem repeats, such as the one and a half repeat of the MAM domain (the mice had a 3 tandem

repeat here). This source also identified the CXXC motif as a self-oligomizer in D1-D3 of the

vWF and MUC2 domains. This implies, as the paper predicts, possible multimers of Zan. The

paper also identifies different splicing patterns, leading to different Zan isoforms.

Tardif and Cormier (2011) find three cell adhesion domains of Zan: MAM (meprin/ A5

antigen/ mu receptor tyrosine phosphotase), mucin, and vWD. They found that mice have a

region of Zonadhesin between D3 and D4 where the last 2 exons of D3 domain have duplicated,

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called the D3 partials. They confirm Hardy and Garber’s (1995) suspicions that Zonadhesin is

utilized as a sperm- egg adhesion protein.

The domains of Zonadhesin are identical in an incredible number of species, with very

high rates of conservation across only weakly related species. The order and number of these

domains, however, differs greatly. These domains have come together from various places in

biology and have combined to perform a precise, important, novel purpose – otherwise there

would be much lower rates of conservation. To find out more about the purpose of Zonadhesin, it

is important to investigate its different types.

ISOFORMS

In this section the research concerning different isoforms of the Zan protein published

between 1995 and 2006 is summarized. Zonadhesin is different from species to species.

Therefore, the intraspecies differences between Zonadhesin molecules in form and functionality

can elucidate the origins of the protein.

Hardy and Garbers (1995) find a purified form of Zan will move through an SDS-PAGE

in nonreducing conditions to show a Mr 150,000 protein, and in reducing conditions, a set of Mr

105,000 and Mr 45,000 subunits, with their disulfide bond disrupted. These are called p105 and

p 45.

Hickox, Bi, and Hardy (2001) confirm Hardy and Garbers’ (1995) assumption that Zan

consists of the p105 and p45 subunits. Because they also found very high Mr isoforms of Zan,

they assumed covalent isoforms were forming (dimers and hexamers). To further substantiate

this claim, the high conservation of CG(L/V)CG in D1-D3 of pig Zan suggested oligomerization,

as this is the role for the sequence in vWF. The p105 and p45 subunits were found to make up

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p150, p300, and p900 isoforms. Of these, the p150 isoform had the highest zona pellucida

binding affinity.

Two years later, Bi, Hickox, Winfrey, Olson and Hardy (2003) identify five isoforms of

Zan in humans. Some bind more effectively to the ZP than others, causing infertility. Some have

differences in glycosylation, which leads to differences n size and shape between different

isoforms and species of Zan, leading to higher binding specificity.

In direct contrast, Gasper and Swanson (2006) identify six isoforms formed by alternate

splicing of exons 41-43 in human Zan. It is unclear whether the disparity is due to a difference in

methodology of technological advances in the field during those three years.

It is clear that Zonadhesin, like many other proteins, has different types, and potential for

great variety in its creation. Monomeric units unite to build isoforms with varying binding

affinities, some much more efficient than others. Evolution creates diversity in the protein

Zonadhesin as it does in all life.

EVOLUTION

The research summarized in this section highlights the sections of the Zonadhesin protein

that have been identified as undergoing or having undergone significant evolutionary changes.

These changes are windows into the function of the protein, because as the structure changes, so

does the function. In this way, the functions of certain parts of the protein are revealed.

Herlyn and Zischler (2005) identify a region of MAM 2 which undergoes positive

selection, meaning that rapid change of amino acids in this region is favored. Study revealed the

30 base pair segment of MAM2 as a probable binding site because of its solubility, which

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indicated it was extracellular and therefore exposed, and theoretically able to bind. Positive

selection at this portion of the protein would cause morphological changes and affect binding.

However it should be noted that this only refers to the possible binding site at the MAM2

domain, and that there may be several others. This study focused only on how a specific site in

the gene changed in relation to itself overtime. These two would soon perform a more intricate

experiment, with more surprising results.

The next year, Herlyn and Zischler (2006a) find that the D3 partials in mouse have faster

paralogue evolution than homologue evolution. The partials are diverging on the codon level,

creating new functions rather than rendering them nonfunctional, suggesting positive selection

and perhaps phylogenetic informativity. They are converging at the small fragment level;

however, the previously stated divergence seems to be offsetting that for now. By comparing the

D3 partials to each other, Herlyn and Zischler (2006a) had opened up a whole new subfield in

the study of the protein. This offered another valuable area of focus for those working in

phylogenetics to search for informativity and divergence.

As segments of the gene are placed under evolutionary pressure, they become areas of

phylogenetic specificity. And as segments of the gene repeat, they allow more information (and,

somewhat counter intuitively, specificity) to be stored, allowing for more phylogenetic diversity.

Yet just as important as the effects of evolution on the protein are the factors which drive its

evolution.

Evolutionary Drive

In this section the research that discusses the factors which drive the evolution of the

Zonadhesin gene, published between 2006 and 2008, is summarized. Different factors affect

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different species to varying degrees, depending on the manner by which mates are selected in

that species. By observing the differences in the driving factors for the protein’s evolution, the

mechanism of function is further revealed.

Herlyn and Zischler (2006a) cite the evolution of the molecular counterpart to

Zonadhesin (the zona pellucida binding proteins) as a driving force to Zonadhesin evolution.

They speculate that, because the zona pellucida binding proteins are constantly evolving, so too

must the D domains of Zonadhesin. Therefore, they conclude that female cryptic choice must

play a large part in species deviation. This drives the focus of the theory of this protein’s

evolution back to a more Darwinian viewpoint (instead of a single protein driving all off

evolution, the protein evolves in tandem with the species). Their research would continue to

follow this trend of speciation informing divergence (rather that divergence of the protein

informing speciation) throughout.

Later the same year, Herlyn and Zischler (2006b) cite sperm competition as a driving

force for Zan evolution. Herlyn and Zischler (2006b) also points out the delicate balance

between evolution to promote diversity in the Zan gene, and the conservation necessary to retain

fertility function. They proposed that species differentiation was spurred on by the accumulation

of positively selected sections. They proposed that the mechanism of this change hinged on

amino acid variation and posttranslational modification changes. The overall conservation they

observed across species, however, led them to postulate that some similarities were necessary in

order to retain the properties which allowed the protein to function.

In a combination of, and to provide reason for, their earlier two studies that same year,

Herlyn and Zischler (2006c) compared body weight dimorphism with Zonadhesin divergence.

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They found that in unimale breeding species, Zonadhesin was evolving at a much slower rate.

They speculated that the level of sperm competition and female cryptic choice is much lower in

these species, and that these factors might drive evolution. Because the level of sexual

dimorphism in weight is high between the genders of these species, they concluded that there

was a negative correlation between the rate of evolution of Zan sequences and the amount of

disparity between average body weight of individuals of opposite sexes in a unimale breading

system.

In a terrific summary of their 2005, and 2006 papers, Herlyn and Zischler (2008) do a

comprehensive job of summarizing the different ways in which different sections of Zan evolve.

They cite concerted evolution as the major factor for change in the mucin-like domains, but drift

and positive selection in the MAM and D domains. This is significant as they assert that positive

selection, as opposed to concerted evolution, is more likely to cause changes in posttranslational

medications, and thus affecting binding sites. Additionally, they point out that negative, not

positive evolution is most common for codon sites. They used moving window correlation

analysis to compare sexual body dimorphism and Zan evolution, concluding that both sperm

competition and female cryptic choice would play a large role in Zan evolution.

It should be noted that every paper here summarized in the sections concerning

Zonadhesin’s evolution has been published by the same two authors. While their work is

extensive, it may be worth considering why they have so completely dominated this subfield of

Zonadhesin research.

Regardless, with a general overview of the structure, variety, and evolution of

Zonadhesin, the most obvious aspect, its function, begs to be explored.

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FUNCTION

This section provides a brief overview of the original research done on the function of the

protein Zonadhesin. It includes the experiment which discovered the protein in 1995 and one of

the more recent experiments, confirming the proteins capacity to transfer species specificity.

As the first to discover the protein Zonadhesin, Hardy and Garbers (1995) accomplished

the task by using the zona pellucida as an isolating medium for the experiment. Because of the

protein’s ability to bind to the egg’s extracellular matrix, and because of the protein’s vWF

(which is a domain commonly used in binding) homologous domains, the researchers proposed

that Zan could be involved in sperm-egg adhesion.

Building, as so many others had, off of Hardy and Garbers (1995), Tardf et al. (2011)

used a gene nullification experiment to test the actual purpose of the protein. This experiment

used male mice that had no Zan protein on their sperm (the mice were, however, fertile, as the

Zan null mouse sperm was able to fertilize mice eggs in vitro). Compared to Zan positive mouse

sperm, the Zan null sperm had higher rates of adhesion to pig, cow, and rabbit zona pellucida,

but not to mouse zonal pellucida. This experiment provided the basis for the assumption of Zan

as the protein which confers species specificity in vertebrates.

This section is exceedingly short for such an important aspect of the protein. This is a

reflection on the lack of research preformed on the function of the protein. Interest seems to be

focused elsewhere, some of the most extensive on binding and localization.

BINDING AND LOCALIZATION

This section provides an overview of the research concerning the binding of the protein

Zonadhesin to the zona pellucida and its localization within the sperm cell prior to and during

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binding. Regions of binding may be of special interest, as their evolutionary patterns can reveal

patterns of evolution within the species. Also, localization of the protein within the sperm reveals

details of the process of fertilization previously unknown.

The first study to focus on localization of Zonadhesin was published by Lea,

Sivashanmugam, and O'Rand (2001) and is the basis of the localization of the Zan proposed by

Bi, Hickox, Winfrey, Olson and Hardy (2003); and Olson, Winfrey, Bi, Hardy and NagDas

(2004); Tardif and Cormier (2011). This study also found that the acrosomal reaction causes the

sperm to lose Zan connectivity, but instead the Zan protein is a part of the acrosomal shroud

where it continues to function. This led the study to propose a post-acrosomal-activation

initiation, contrary to the belief held at the time that the protein was involved in targeting the egg

zona pellucida.

Two years later, Bi, Hickox, Winfrey, Olson and Hardy (2003) confirmed the localization

of the Zan proposed by Lea, Sivashanmugam, and O'Rand (2001) and heavily references Hickox,

Bi, and Hardy (2001) in the isoforms formed by pig Zan during processing. Bi, Hickox, Winfrey,

Olson and Hardy (2003) adds on to the study in Hickox, Bi, and Hardy (2001) by performing

detailed analysis of the glycosylation types of the isoforms, and where each isoforms tended to

be located in the sperm. By finding the Zan isoforms were located in the acrosome; they were

able to conclude that Zan- Zona pellucid interaction likely happened during exocytosis of the

acrosome.

Also in agreement, Olson, Winfrey, Bi, Hardy and NagDas (2004) confirmed the

localization of the Zan proposed by Lea, Sivashanmugam, and O'Rand (2001) and identifies the

process and specific localization of Zan within the acrosome of the sperm. Zan is first distributed

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equally across the domains of the inner and outer acrosomal membranes in round spermatids.

Zan is then limited to the outer acrosomal membrane, and after sperm maturation, is taken into

the acrosomal matrix itself. The study suggests Zan may have a structural role in the matrix as

well.

Focusing more on the proteins binding ability, Herlyn and Zischler (2005) deduced a

potential binding site in the MAM 3 domain of the Zan protein due to the higher levels of

posttranslational modification. These posttranslational modification changes were caused by

positive selection at a specific site, at the level of the amino acids. These modifications resulted

in predicted motifs that were highly variable, giving the protein more ability to specify binding

sites.

Following suit, Gasper and Swanson (2006) sequenced exons of Zan to look for sites

under positive selection. In a sample which consisted of twelve primate species, including

humans, they found that VWD2, MAM, and Mucin domains of Zan all had exons under

significant levels of positive selection, enough to indicate that these regions could be sites

involved in zona pellucid binding in the mature protein.

Tardif and Cormier (2011) confirm the localization finding of Bi, Hickox, Winfrey,

Olson and Hardy (2003) and confirms the localization of the Zan proposed by Lea,

Sivashanmugam, and O'Rand (2001). They also add that Zan can first be detected in round

spermatids. The Zan protein is later modified and moved to the acrosome of the sperm. In

addition, they postulate that Zan is not exposed during AR, but rather during capacitation and

that it may be reversible, further expanding on the method of exocytosis of the protein proposed

by Lea, Sivashanmugam, and O'Rand (2001).

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This section in particular shows the cooperativity in the most active aspects of this field

of research. Not a single paper could have stood on its own – each needed pieces of the other’s

research to make sense of their own. And together they were able to propose novel mechanisms

of localization and activation.

CONCLUSION

In summary, the scholarship of Zonadhesin is far from complete. The sequence is known,

as are the domains, and variations thereof. Which isoforms bind well, and how they occur in

vivo, is well agreed upon. The function is presumed to be so straight forward (conferring species

specificity), that it seems to be pushed out of the spotlight of the research.

But research on the evolution of the protein of itself, if not the factors that drive it, is far

from completed. Every site, whether it binds to the zona pellucida of the protein or not, which

identified as experiencing some kind of evolutionary pressure is a tool for phylogenetic analysis

of species. And the binding and localization of the protein is still a hotly debated subject, further

complicated by the fact that fertilization itself is not fully understood at the molecular level.

Bibliography

A Sperm Membrane Protein That Binds in a Species-specific Manner to the Egg Extracellular Matrix Is Homologous to von Willebrand Factor

Daniel M. Hardy‡ and David L. Garbers§ From the Department of Pharmacology and Howard Hughes Medical Institute, University of

Texas Southwestern Medical School, Dallas, Texas 75235-9050

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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 44, Issue of November 3, pp. 26025–26028, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Expression and genomic organization of zonadhesin-like genes in three species of fish give insight into the evolutionary history of a mosaic protein Peter ND Hunt1, Michael D Wilson1, Kristian R von Schalburg1, William S Davidson2 and Ben F Koop* Address: 1Centre for Biomedical Research, University of Victoria, Victoria, British Columbia V8W 3N5, Canada and 2Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada Email: Peter ND Hunt - phunt@uvic.ca; Michael D Wilson - mdwilson@uvic.ca; Kristian R von Schalburg - krvs@uvic.ca; William S Davidson - william_davidson@sfu.ca; Ben F Koop* - bkoop@uvic.ca * Corresponding author Published: 22 November 2005 BMC Genomics 2005, 6:165 doi:10.1186/1471-2164-6-165 Received: 21 July 2005 Accepted: 22 November 2005 This article is available from: http://www.biomedcentral.com/1471-2164/6/165 © 2005 Hunt et al; licensee BioMed Central Ltd.

MOLECULAR BASIS OF CELL AND MOLECULAR BIOLOGY: Heterogneous Processing and Zona Pellucida Binding of Pig Zonadhesin John R. Hickox, Ming Bi and Daniel M. Hardy

J. Biol. Chem. 2001, 276:41502-41509 doi: 10.1074/jbc.M106795200 originally published online August 28, 2001 Identification of a positively evolving putative binding region with increased variability in posttranslational motifs in zonadhesin MAM domain 2 Holger Herlyn *, Hans Zischler Institute of Anthropology, University of Mainz, Colonel-Kleinmann-Weg 2 (SB II) D-55099, Germany Received 12 October 2004; revised 25 February 2005 Available online 31 May 2005 Molecular Population Genetics of the Gene Encoding the Human Fertilization Protein Zonadhesin Reveals Rapid Adaptive Evolution Joe Gasper and Willie J. Swanson The American Journal of Human Genetics Volume 79 November 2006 Processing, localization and binding activity of zonadhesin suggest a function in sperm adhesion to the zona ellucid during exocytosis of the acrosome Ming BI*, John R. HICKOX*, Virginia P. WINFREY†, Gary E. OLSON†and Daniel M. HARDY*1 *DepartmentofCellBiology&Biochemistry,TexasTechUniversityHealthSciencesCenter,3601FourthStreet,Lubbock,TX79430,U.S.A.,and†DepartmentofCellBiology, VanderbiltUniversitySchoolofMedicine,Nashville,TN37232,U.S.A.

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Role of zonadhesin during sperm–egg interaction: a species-specific acrosomal molecule with multiple functions Steve Tardif* and Nathaly Cormier Reproductive and Developmental Biology Group, Maternal and Child Health Sciences Laboratories, Centre for Oncology and Molecular Medicine, Division of Medical Sciences, Ninewells Hospital, University of Dundee, DD1 9SY, Dundee, UK *Correspondence address. Email: s.tardif@dundee.ac.uk Submitted on April 15, 2011; resubmitted on May 14, 2011; accepted on May 16, 2011 Molecular Human Reproduction, Vol.17, No.11 pp. 661–668, 2011 Advanced Access publication on May 20, 2011 doi:10.1093/molehr/gar039 SEQUENCE EVOLUTION OF THE SPERM LIGAND ZONADHESIN CORRELATES NEGATIVELY WITH BODY WEIGHT DIMORPHISM IN PRIMATES Holger Herlyn1,2 and Hans Zischler1,3 1Institute of Anthropology, University of Mainz, Colonel-Kleinmann-Weg 2 (SB II), D-55099, Germany 2 doi:10.1111/j.1558-5646.2007.00035.x Received August 24, 2006 Accepted October 9, 2006

Sequence evolution, processing, and posttranslational modification of zonadhesin D domains in primates, as inferred from cDNA data Holger Herlyn*, Hans Zischler Institute of Anthropology, University of Mainz, Colonel-Kleinmann-Weg 2 (SB II), D-55099 Mainz, Germany Received 27 September 2004; received in revised form 3 May 2005; accepted 2 June 2005 Available online 26 September 2005 Received by W. Makalowski Isabel A. Lea,2 Perumal Sivashanmugam, and Michael G. O’Rand Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 BIOLOGY OF REPRODUCTION 65, 1691–1700 (2001)

Zonadhesin D3-Polypeptides Vary among Species but Are Similar in Equus Species Capable of Interbreeding1 Steve Tardif,6 Heidi A. Brady,7 Kelly R. Breazeale,3,7 Ming Bi,4,6 Leslie D. Thompson,7 Jason E. Bruemmer,8 Laura B. Bailey,5,7 and Daniel M. Hardy2,6 Department of Cell Biology & Biochemistry,6 Texas Tech University Health Sciences Center, Lubbock, Texas Department of Animal and Food Sciences,7 Texas Tech University, Lubbock, Texas Department of Animal Sciences,8 Colorado State University, Fort Collins, Colorado BIOLOGY OF REPRODUCTION 82, 413–421 (2010) Published online before print 30 September 2009. DOI 10.1095/biolreprod.109.077891 Zonadhesin Is Essential for Species Specificity of Sperm Adhesion to the Egg Zona Pellucida Receivedforpublication,March12,2010,andinrevisedform,May26,2010 Published,JBCPapersinPress,June7,2010,DOI10.1074/jbc.M110.123125 SteveTardif‡1,MichaelD.Wilson§1,2,RebeccaWagner§,PeterHunt§,MarinaGertsenstein¶,AndrasNagy¶, CorrinneLobe,BenF.Koop§,andDanielM.Hardy‡3 Fromthe ‡DepartmentofCellBiologyandBiochemistry,TexasTechUniversityHealthSciencesCenter,Lubbock,Texas79430-6540, the

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§DepartmentofBiology,CentreforBiomedicalResearch,UniversityofVictoria,Victoria,BritishColumbiaV8P5C2,Canada,the ¶SamuelLunenfeldResearchInstitute,MountSinaiHospital,Toronto,OntarioM5G1X5,Canada,andtheMolecularandCellular BiologyDivision,SunnybrookHealthSciencesCentre,Toronto,OntarioM4N3M5,Canada THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 32, pp. 24863–24870, August 6, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A