Human Reproduction Update, Vol.15, No.2 pp. 237– 247, 2009 Advanced Access publication on December 24, 2008 doi:10.

1093/humupd/dmn060

Proprotein convertase subtilisin/kexin type 4 in mammalian fertility: a review
Charles Gyamera-Acheampong1,2 and Majambu Mbikay1,2,3,4
1 Chronic Disease Program, Ottawa Health Research Institute, 175 Parkdale Avenue, Ottawa, ON, Canada K1Y 4E9 2Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada 3Division of Endocrinology and Metabolism, The Ottawa Hospital, University of Ottawa, Ottawa, ON, Canada 4

Correspondence address. Tel: þ1-613-798-5555; Fax: þ1-613-761-4355; E-mail: mmbikay@ohri.ca

table of contents
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Introduction Methods PCSK4 is a member of a family of activating endoproteinases Biochemical properties of PCSK4 PCSK4 in reproduction: lessons from the PCSK4 deficient mouse Clinical implications of PCSK4 for human fertility Conclusion

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background: Proprotein convertase subtilisin/kexin type 4 (PCSK4), also known as proprotein convertase 4, belongs to a family of endoproteinases involved in the proteolytic conversion of secretory precursor proteins to their active forms. Its amino acid sequence is highly conserved in mammals, an indication of its biological importance. methods: We have searched PubMed and molecular biology databases for information relating to the structure, expression and biological functions of PCSK4. results: PCSK4 is predominantly expressed in male germ cells and located on the plasma membrane overlying the acrosome of sperm.
It is also present in ovary and placenta. Inactivation of its gene in mouse does not alter spermatogenesis, but renders sperm incapable of fertilizing oocytes. This incapacity results in part from sperm susceptibility to a premature acrosome reaction and their reduced ability to bind to the zona pellucida. In female mice, a lack of PCSK4 causes subfertility associated with impaired folliculogenesis. In addition, this enzyme has been shown to stimulate the invasiveness of human placental trophoblasts in culture, suggesting that it may facilitate placentation in vivo.

conclusions: PCSK4 appears to be a crucial enzyme for reproduction. Alterations of PCSK4 expression or activity could be the underlying cause of some unexplained cases of human infertility. Conversely, inactivation of this protease represents a potential strategy for non-hormonal contraception.
Key words: PCSK4/PC4 / proprotein convertase / protease / reproduction / fertility

Introduction
Proprotein convertase subtilisin/kexin type 4 (PCSK4), also known as proprotein convertase 4 (PC4), belongs to a nine-member family of calcium-dependent serine endoproteinases involved in the proteolytic activation of secretory precursor proteins to their bioactive forms (Van de Ven et al., 1993; Seidah et al., 1994; Steiner, 1998; Seidah and Chretien, 1999; Seidah and Prat, 2002). Its transcripts and protein are primarily found in testicular germ cells and sperm

(Nakayama et al., 1992; Seidah et al., 1992; Torii et al., 1993; Gyamera-Acheampong et al., 2006), but its transcripts are also detectable in ovary (Tadros et al., 2001), placenta (Qiu et al., 2005) and oocytes (St. Germain et al., 2005). Consistent with its distribution in reproductive tissues, inactivation of its gene in mice causes male infertility to males and subfertility to females (Mbikay et al., 1997). The high conservation of the PCSK4 sequence and its expression pattern among tetrapods suggests that this enzyme may have played a critical role for reproductive success during evolution. Yet much remains to

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a P domain.. SPC6)...... Grozdanov et al... These include hormones.. Based on structural similarities to yeast and bacterial proteases... 1)...... 2008)...... 1999.. SPC3)....... PC3.. Seidah et al.. the C-terminally exposed basic residue is removed by a carboxypeptidase (CP) unless preceded by a Pro. fes upstream protein.. 1993... b X stands for any amino acid except Cys. Except for PCSK3 and PCSK6.... SPC1). a Furin. 2008).. PCSK7 (PC7. Often.. Glu and Val (Espenshade et al.. The cleavage specificity of the three PCSK clans is shown in Table I..... subtilisin/kexin isozyme 1.238 be elucidated about its natural substrates and their roles in reproductive physiology. S1P.... palmitoylation (van de Loo et al. SPC7)... The enzymatic activities of PCSKs are variably dependent on calcium levels and pH in the successive compartments of the secretory pathway. and a variable C-terminal domain (Fig. 2000). a conserved catalytic domain... After cotranslational removal of the signal peptide.oxfordjournals... A Gly exposed by CP can be converted to an amide group by peptidylglycine-alpha amidating monooxygenase (PAM)..... They are transcribed in all nucleated cells in different combinations and amounts.. PCSK4 (PC4. they are further subdivided into kexin-like..... V)-Z#Xc V-F-A-Q#S-I-Pd .. ‘#’ for cleavage and ‘n’ for position P2. Mature PCSK9 has no known physiological substrate. 1995.. anchored to or associated with the plasma membrane. PC8.. Other post-translational modifications known to occur include glycosylation (Benjannet et al. according to the proposed unifying nomenclature (Fugere and Day..... Aliases found in the scientific literature are provided in Table I.. Ensembl.. These algorithms are specified in the text. Mayer et al..... The final destination of the activated PCSK is variable: the enzyme could be retained in the Golgi or the TGN. neural apoptosis-regulated convertase 1.. 1997.. 2008). Seidah and Prat. RNA or DNA sequences across species as well as for annotations of interest. Cys.. 2005).. this transcription is considered ubiquitous. 2004... d This is the site of autocatalytic cleavage of proPCSK9 after its prodomain V-F-A-Q#S-I-P (Seidah et al.. lymphoma proprotein convertase.. Creemers et al.... 2006) and C-terminal truncation (Vindrola and Lindberg. PCSK6 (PACE4...org/ by guest on November 27. PCSK3 (furin... Expasy) for PCSK4 protein.. Seidah and Chretien.. 1996. 1997) or polyadenylation (Ftouhi et al... sulfation (Benjannet et al. 1997..... SPC... 1)....... 2002.. site-one protease.. Benjannet et al.. 1992). 2002). PCSK8 (SKI-1...... 1997).... PCSK2 (PC2. PACE... phosphorylation (Jones et al.. 2002. 1999.. paired basic amino acid cleaving enzyme... LPC.... 1994). Finally. we discuss the possible usefulness of PCSK4 as a marker of fertility and target for nonhormonal contraception. we sum up current knowledge on PCSK4 biology: we briefly describe biochemical properties common to all PCSKs before expanding on particularities of PCSK4 structure... P4.. Toure et al.. we searched genetics and molecular biology online databases (NCBI.... 1999. Among proteases.. subtilisin-like proprotein convertase... PC6... 1994). splicing (Mbikay et al... 2002). LPC. S1P) PCSK9 (NARC-1) Cleavage specificity (R/K)n-X-K/R#Xb R/K-X-(L. PCSKs are encoded by genes of different exon numbers and introns sizes..... these genes map on different chromosomes...... PCSK4 is a member of a family of activating endoproteinases In this review... Seidah and Chretien.. 1997. 2006). PCSKs of the other two clans regulate lipid metabolism: pCSK8 is one of the two endoproteases that mediate the release of sterol regulatory element binding proteins (SREBPs) from ER membranes.. widespread or restricted (Seidah and Chretien. 1999.. global transcriptome analysis using DNA microarrays and sequencing of cloned expressed sequence tags (ESTs) have shown that expression of all PCSK genes spreads across tissues and cell types.. 2006. stored in neuroendocrine secretory granules.... As demonstrated experimentally for PCSK3 (Anderson et al. HapMap... 1999. 1999).. the resulting proPCSK undergoes maturation by autocatalytic cleavage between the pro and the catalytic domains. I. 2002).. neuropeptides and growth factors as well as cell surface receptors and integrins (Nakayama. In this review.. where # stands for a number).. NARC-1. where a secondary autocatalytic cleavage within the propeptide dissociates the complex. we used online algorithms for comparative analysis of conserved potentially functional motifs in PCSK4 DNA and protein sequences.. Most physiological products of precursor processing by kexinlike PCSKs are implicated in cell signaling as ligands or receptors. recycled into endosomes.. PCSKs are biosynthesized in the endoplasmic reticulum (ER) as multidomain preproproteins consisting of an N-terminal signal peptide followed by a pro domain.. lastly... it is generally believed that the propeptide remains attached to the mature enzyme until the complex reaches the trans-Golgi network (TGN). Tsuji et al... P6 or P8.... In addition. all members of this family of enzymes are called proprotein convertase subtilisin/kexin type # (PCSK#. 2012 Table I Nomenclature of proprotein convertases subtilisin/kexin-like Clans Kexin-like Pyrolysin-like Proteinase K-like Adopted names (aliases)a PCSK1 (PC1.... expression and enzymatic activity.. SKI-1. 1993. within a cleavage motif that is also recognized by the fully activated enzyme in the primary sequence of its physiological substrates (Muller and Lindberg... PCSK5 (PC5... or secreted or shed into the extracellular milieu (Seidah and Prat. Seidah and Prat.. 2003.. PACE. PCSKs belong to the family of serine proteases of the subtilisin subtype. Seidah et al.. allowing nuclear translocation of these master Downloaded from http://humupd.. rendering the enzyme fully active (see Fig. c Z stands for any amino acid except Pro.. Seidah and Prat. 1994... After cleavage.... However... SPC4)... ‘/’ for ‘or’... Dewpura et al. van de Loo et al. .. the transcription produces one major mRNA form and several minor ones resulting from the use of alternative sites of transcription start (Ayoubi et al.... Based on the relative mRNA abundance. 2000). pyrolysin-like or proteinase K-like clans (Table I) (Siezen and Leunissen.. Seidah and Chretien. proprotein convertase.. SPC2). PC.. we then assess the contribution of this enzyme to reproduction as learned from the phenotypic characterization of PCSK4-null mice.. SPC5).. Gyamera-Acheampong and Mbikay Methods Relevant publications were searched in PubMed using as keywords the various names of PCSK4 and its related enzymes...

reducing plasma LDL uptake by these cells (Horton et al. Creemers and Khatib. which in mouse testis represent 10% of all Pcsk4 transcripts (Seidah et al. S1). 2008)..oxfordjournals. 2006). 2005). it also carries a transmembrane (TM) domain. In rodents. 1992. 2007).. effectively reducing the level of translatable mRNA by making the transcripts susceptible to nonsense-mediated decay (Lewis et al. Mbikay et al.. Underneath the prodomain and its junction to the CAT domain are given the sequence around the primary and secondary basic cleavage sites in proPCSK4 (capital characters) and proPCSK3 (small characters). 2003). 1992... The open reading frame (ORF) of these alternate mRNAs encode proteins with various deletions or insertions when compared with the major isoform (Seidah et al. The variable C-terminal (CT) domain influences intracellular localization. Besides the fact that during spermatogenesis PCSK4 transcripts first appear at the pachetene spermatocyte stage and later disappear at the elongated spermatid stage. it also shows a beta sandwich fold typical of galactose binding domain-like which may mediate attachment to carbohydrates. Immunoreactive PCSK4 protein is present in mouse male germ cells and on the surface of epididymal sperm (Gyamera-Acheampong et al.. 1995).. 2006).. This cluster of regulatory elements is also found in the upstream region of rat and macaque PCSK4 genes. 1994). Note the absence of a basic secondary cleavage site in the prodomain of PCSK4.. 2005). The catalytic domain (CAT) carries the Asp– His – Ser catalytic triad characteristic of serine proteinase. Fig. Downloaded from http://humupd. which extends from the prodomain to the P domain (Fig. phospholipids or membrane receptors. 2005). 2).. In female mice.. This high .... 2006.org/) reveals that PCSK4 transcripts are most abundant in reproductive organs. the transcripts are detected by reverse transcriptase –polymerase chain reaction (RT –PCR) in mouse ovarian macrophage-like cells (Tadros et al. Overall. Alignment of the 2 kb sequence upstream of the transcription start site of the human and mouse gene reveals a 600 bp internal domain of high sequence similarity containing several conserved binding sites for transcription factors (Supplementary data. in – out recycling as well as protein – protein interactions. rodents and frogs reveals a remarkable conservation of its sequence. The prodomain (PRO) serves as intramolecular chaperone and transient inhibitor. Seidah et al. this distribution strongly suggests that the function of PCSK4 is linked to reproductive processes. The physiological significance of these isoforms is unclear. 2000. 1992. The P(rotease) domain influences Ca2þ and pH dependence. Gyamera-Acheampong et al. 1998). they are primarily found in germ cells.. For most PCSKs. The signal peptide (sp) directs the nascent precursor toward the secretory pathway. 2007. Putative N-glycosylation sites are indicated by buds. particularly in the testis. In adult frog. Production of alternative mRNA isoforms has been interpreted to be a mechanism of extending the informational content of the genome and expanding the proteome (Maniatis and Tasic. 2008). oocytes and early embryos (Nelsen et al. followed by brain. Organizationally and sequence wise. Torii et al. 1995). Essalmani et al.PCSK4 protease and fertility 239 Figure 1 Schematic representation of human PCSK4 domains.. nothing is known about the regulation of PCSK4 gene transcription.. the PCSK4 gene is more homologous to PCSK3 gene than to any other gene of the PCSK family (Mbikay et al. Their presence has also been demonstrated in human placenta and placental cell lines (Qiu et al... 2008). Mbikay et al. 2001) as well as in unfertilized and fertilized oocytes (St. It may also play a regulatory role when it introduces a premature nonsense codon in the ORF. testis and ovary are the primary sites of expression of these transcripts. the PCSK4 gene is transcribed into a major mRNA of 3 kb and several shorter isoforms resulting from alternate splicing.gnf. 2012 transcriptional activators of genes involved in lipid biosynthetic pathways (Horton et al. 2003. 1993. mostly in spermatocytes and round spermatids (Nakayama et al. 15 exon gene located on human chromosome 19 (symbol: PCSK4) and mouse chromosome 10 (symbol: Pcsk4) (Mbikay et al. Bergeron et al. 2005) as well as in human endometrium (Freyer et al. Molecular analysis of precursor processing in PCSK-null mice has revealed both substrate specificity and redundancy among the convertases (Creemers and Khatib. Biochemical properties of PCSK4 Structure and expression PCSK4 is the product of a 9 kb. PCSK9 promotes the degradation of the receptor for low density lipoprotein (LDL) in hepatocytes. In this organ.. 2002).. including fertilization and early development. 2008.. Lopez. 1992. One of these factors. 1994). An alignment of the PCSK4 protein sequence of humans. Germain et al. 2002). The expression level and pattern of this gene may be determined by interactions of multiple factors over this domain. Global profiling of mouse and human transcriptome using DNA microarrays (published online at http://symatlas...org/ by guest on November 27. the nuclear deformed epidermal autoregulatory factor-1 (DEAF-1) related factor (NURD) is reportedly expressed at high levels in male germ cells (Huggenvik et al. as well as in human placenta (Qiu et al.. genetic nullizygosity causes biological anomalies ranging from embryonic lethality and developmental defects to metabolic disturbances and infertility (Taylor et al. Scamuffa et al.

1997.ibcp. mouse (Mus musculus) and frog (Xenopus laevis) preproPCSK4 sequences are Q6UW60.oxfordjournals.. RR#) (Remacle et al. Its optimal activity is observed near Downloaded from http://humupd. this domain also carries a transmembrane domain. PCSK4 enzymatic activity Despite the inefficient maturation and trafficking of proPCSK4 in transfected cells. which appear to have rapidly evolved during speciation (Torgerson et al. The transmembrane membranes are underlined. it has been possible to detect its activity in the culture media of these cells (Basak et al. 2006). like proPCSK2 (Muller et al. When transduced into somatic cells in culture.fr/). 1997. In most PCSKs. 2008).ncbi. 2012 Figure 2 Alignment of tetrapod PCSK4.nlm. The nature of this association remains to be elucidated. an indication that the latter may be subject to further proteolytic processing. The biosynthesis and trafficking of PCSK4 in the secretory pathway has not been elucidated. it is still unclear how mature PCSK4 becomes fully activated since its prodomain lacks a Gyamera-Acheampong and Mbikay recognizable internal secondary site (see Fig. The catalytic domain is delimited with blue brackets. In primates.. The active residues in the catalytic domain are indicated with dots. This molecular mass is smaller than predicted for mature mouse PCSK4 (62 kDa). 2008). to partially purify the enzyme from these media (Basak et al. most often after paired basic residues (KR#. Mbikay et al.. Cleavage sites leading to removal of the signal peptide and the prodomain are indicated with a filled and an open arrow. Rodent PCSK4 lacks a transmembrane domain in its C-terminal domain. cleavage at this site rids the mature enzyme of the inhibitory propeptide generated by the primary cleavage at the demarcation between the pro and the catalytic domains (Anderson et al. 1999. 2002). The C-terminal domain is the least conserved.gov/) accession numbers for humans (Homo sapiens). 1999. it is detectable by immunoelectron microscopy at the surface of intact mouse sperm (Gyamera-Acheampong et al. respectively. Residues that are conserved in all four species are written in red. 2006). 2004) and. 2002). EDL89 295.. 2008). NCBI database (http://www. making PCSK4 a type-1 membrane protein.org/ by guest on November 27. It suggests that PCSK4 gene was positively selected during evolution because of its biological importance. 1997. this zymogen requires the assistance of a specific chaperone to efficiently navigate through the secretory pathway.nih. most likely to C-terminal truncation.. The sequences were aligned using the MULTALIN online algorithm (http://pbil. NP_032819 and AAW83 023. Yet.. . X ¼ any residue) (Basak et al. The Arg-Gly-Asp (RGD) motif that interacts with integrin is boxed. 1). PCSK4 from mouse testis and sperm migrates on western blots with an electrophoretic mobility of 54 kDa (Mbikay et al. respectively.. in blue. rat (Rattus norvegicus). but it cleaves uniquely better after a single Arg preceded by a Lys at P4 (KXXR#. Like all kexin-like PCSKs. Furthermore. most of it is retained in the ER as proPCSK4 (our unpublished data). more recently.. Gyamera-Acheampong et al. 2004. 2001.240 conservation is in contrast with many mammalian sperm-specific proteins.. 2001). Tadros et al.. besides being longer and richer in Cys residues. It is possible that.. those conserved in two or three. recombinant PCSK4 was shown in vitro to cleave synthetic peptide substrates after an Arg in a basic sequence context..

......... 2001) (Mbikay et al... 1997......... Downloaded from http://humupd..... PCSK4-null sperm are capacitated faster and undergo near complete acrosome reaction in the presence of amounts of zona pellucida that are ineffective on normal sperm...........oxfordjournals... 2004) (Li et al........ 2006). propituitary adenylate cyclase-activating peptide (proPACAP)......... (Shioda et al. PCSK4 in reproduction: lessons from the PCSK4 deficient mouse Physiological defects The production of a PCSK4-null mouse by disruption of its genomic locus (Mbikay et al. pronerve growth factor (Parvinen et al..... 1990... (iii) its proteolytic processing must be impaired in PCSK4-null mice and (iv) its deficiency must cause an infertility phenotype...... .... n ¼ 24) (Mbikay et al... 1996)...8 pup/litter. 1997)......... No difference between wild-type and null mice was observed in seminiferous epithelium morphology and sperm number or motility... The fertility phenotype of this mouse is summarized in Table II.. insulin-like growth factor-1 (IGF-1) receptor Table II Fertility phenotype of PCSK4-null mice Defects Physiological defects Male mice Infertility Accelerated sperm capacitation Susceptibility to premature sperm acrosome reaction Reduced sperm hyperactivation Reduced sperm binding to oocytes Inability to fertilize oocytes Female mice Subfertility Reduced folliculogenesis Reduced ovulation Molecular defects Male mice Reduced levels of spermatid PACAP-regulated MAPKs Lack of testicular PACAP peptides Female mice Lack of ovarian PACAP peptides (Li et al.......... null male congenics were infertile and their sperms were unable to fertilize oocytes in vitro (Gyamera- In testis Male germ cells biosynthesize several precursor proteins that are known to need PCSK-mediated limited endoproteolysis to become active...... Female phenotype Female mice were found to be mildly subfertile.. 2006) (Mbikay et al.......... 2000a. 2006) References ..... hyperactivation of PCSK4-null sperm after capacitation was half that of wild type cells... This subfertility appears to be caused in part by impaired folliculogenesis (Tadros et al.. 1998.... 2006)....org/ by guest on November 27. resulting in the release of the most active IGF21 – 67 after carboxypeptidase (CP)-mediated removal of the terminal basic residue (Qiu et al.......... 1997).. 1999). Ex vivo.. Gyamera-Acheampong et al.... they also bind less efficiently to intact oocytes (Gyamera-Acheampong et al... Null male mice were severely subfertile (25% productive mating..... 1999). 2000a.... 1997) (Tadros et al. b) (Mbikay et al. Seidah et al. Li et al... exhibiting a 23% reduction in productive mating and 65% reduction in litter size relative to heterozygous littermates (Mbikay et al.. 2001)....... 0.. PCSK3 or PCSK7.. Search for potential substrates The availability of the PCSK4-null mouse has also provided the opportunity search for physiological substrates of this enzyme in gonads.. 1997)..... which cleave only within the RLRR104 #FF motif..PCSK4 protease and fertility 241 Acheampong et al. 1994. neutral pH and at about 2 mM Ca2þ (Basak et al..... 1998)... PCSK4 was also shown to process a recombinant 156 amino acid long human proinsulin-like growth factor 2 (proIGF2) within the KSER68 #DV sequence. 2005). This site is not recognized by PCSK1. 1997) has provided significant insights into the biological functions of this enzyme. 1997) (Gyamera-Acheampong et al. generating a larger IGF21 – 101 (Duguay et al.......... b) (Li et al. Breslin et al. 2012 Male phenotype The original null mice were of mixed 129Sv/C57BL/6J (B6) genetic background. In vitro fertilization (IVF) of normal oocytes with mutant sperm resulted in less embryos developing to the blastocyst stage (Mbikay et al.... However.......... 2006) (Gyamera-Acheampong et al...... Counted among these proteins are the following: proenkephalin (Kew et al. This search has been guided by the following criteria: (i) the substrate must be a secretory precursor protein activated by limited proteolysis at sites recognizable by PCSK4...... 2006) (Gyamera-Acheampong et al........ (ii) the precursor must be expressed in the same cells as PCSK4...... When the mutation was backcrossed into the B6 genetic background.. 1993)......... 2001) (Tadros et al... 1992.. 2006) (Gyamera-Acheampong et al.

Downloaded from http://humupd. 1996. 2000a.. 2004.. Bigler et al.. b) and show signs of impaired folliculogenesis (Tadros et al.. inactivation of ADAM2 and ADAM3 genes causes a male infertility phenotype (Cho et al. 2001).. Interestingly. synthetic peptides encompassing some of these sites have been shown to be susceptible to PCSK4-catalyzed hydrolysis in vitro (Basak et al.. Furthermore. Like in PCSK4-null mice.. 2000).oxfordjournals.. steroidogenesis remains unchanged in PACAP-null mice. Shamsadin et al. in wild-type counterparts.. 2008). PACAP38 and PACAP27. 2000a.. 1997.. an early second messenger in the cascade of signaling events that lead to changes in gene expression or cellular metabolism (Vaudry et al. altered signaling is suggested by the reduction of PACAP. lack of placental IGF2 due to disruption of the placenta-specific promoter of the Igf2 gene leads to reduced placental diffusional exchange of nutrients and smaller fetuses (Sibley et al. the only substrate unambiguously identified as a PCSK4 substrate in male germ cells is proPACAP. 2001). 2000.. Ex vivo. 2000). These observations suggest that a strict stoichiometry of interacting proteins may be necessary for the fertilization competence of sperm cells. These ADAMs are biosynthesized in male germ cells as type-1 transmembrane precursor proteins and undergo a cascade of testicular and post-testicular proteolytic cleavages to their acrosomebound forms (Linder et al. proIGF2 is the major form of circulating IGF2 and its conversion to smaller active forms increases during . Its active products. Moreover. More investigation is needed to determine whether and how the reduction of PACAP-regulated MAPKs causes the fertilization defects observed in PCSK4-null mice. PACAPs induce increased production of cAMP. PACAP-null female mice show normal ovarian morphology and ovulation. if impaired proteolytic activation of specific precursor proteins is the main cause of infertility in male PCSK4-null mice. it decreases to the level detected in the mutant mouse. 2004). This feedback transcriptional up-regulation of substrate gene expression has also been observed for hypothalamic growth hormone-releasing hormone in the PCSK1-null mouse (Zhu et al. 1994). In the PCSK4-null mouse. 2004).. this phenotype includes reduced sperm ability to bind to zona pellucida. 2006).. 2008). Blobel. 2000a. 1998. Blobel. PACAP peptides are produced during spermiogenesis (Shioda et al. 1999. sperm –oocyte membrane fusion is normal. Depuydt et al. ProPACAP To date. 1997. Gyamera-Acheampong and Mbikay ADAMs: Members of the ADAM family.type counterparts. 2000a. 2003). By 15 months of age. which includes disturbances of thermogenesis. 1996) and members of the a-disintegrin-and-metalloproteinase (ADAM) family of proteins (Wolfsberg and White. Interestingly..242 (Lehmann et al. known to interact with integrins. One could speculate that PCSK-mediated cleavage at these upstream sites in vivo might expose the disintegrin domain and contribute to its functionality. In mouse... but also by PCSK7 which is also highly expressed in these cells (Bergeron et al. In contrast. b). Therefore. but their mechanism of action is unclear (Almog and Naor. It probably reflects an obligatory and exclusive enzymatic link between a PCSK and its most physiologically important substrates.. 2012 In ovary ProPACAP: The ovaries of PCSK4-null female mice are also devoid of PACAP peptides (Li et al. ADAM2 or ADAM3 deficiency is accompanied by changes in the expression of other sperm proteins including ADAM1 (Nishimura et al. In mouse. Aberrant redistribution or loss of sperm surface proteins has also been observed following disruption of the gene for testicular chaperone calmegin (Ikawa et al. However. in particular ADAM1 (fertilin a). Takahashi et al. In rat. Synthetic peptides containing this motif can block sperm –oocyte fusion in IVF assays (Yuan et al. PCSK4 deficiency is associated with a 20-fold increase in testicular proPACAP mRNA (Li et al. PCSK4 has been shown to stimulate placental trophoblast cell migration by converting proIGF2 to its most active form (Qiu et al. 1998. 2000).. ADAM2 (fertilin b) and ADAM3 (cyritestin).. Their receptors are found in Leydig cells and spermatids. 2002) and pancreatic islet proglucagon in the PCSK2 deficient mouse (Furuta et al. IGFs promote fetoplacental growth (Fowden..regulated mitogen activated protein kinase (MAPK) expression in spermatids (Li et al. Evans et al. Nishimura et al. 1993. It remains to be verified whether this lack protects these mice against age-induced testicular degeneration. 2001). b). this activation must depend exclusively on this convertase. are undetectable in PCSK4-null mouse testis (Li et al. are potential substrates for PCSK4.org/ by guest on November 27. 1999). Like in testis. 1999. Surviving male mutant mice are fertile.. the seminiferous epithelium of old mutant mice remains largely devoid of signs of degeneration observable in same age wild-type mice (Lacombe et al. b). 2004). Sherwood et al. suggesting an enzyme– substrate obligatory link in this organ too. 1997) or angiotensin converting enzyme (Yamaguchi et al. There is currently no evidence that lack of PCSK4 leads to impaired processing of testicular ADAMs. PCSK4 deficiency is associated with a significant increase in proPACAP mRNA (Li et al. 2000) and protein synthesis in spermatocytes (West et al.. The fertility of PACAP-null male mice implies that lack of PACAP peptides in male germ cells and sperm is probably not the primary cause of infertility in PCSK4-null mice. PACAP-null mice show a complex phenotype. lipid and carbohydrate metabolism as well as neuronal development (Gray et al. Lum and Blobel. At 4 months of age. 2001. Of particular interest are sites located closely upstream of the CRXXXXCDXXEXC disintegrin motif. but the mating success is only one-fifth of wild-type controls due to an implantation defect (Isaac and Sherwood. Naz and Padman. 2001). 1995. indicating that neither ADAM plays a unique role in this process. 2001. these observations indicate that PACAP deficiency delays of testicular deterioration associated with aging.. whereas... 1998. suggesting that lack of PACAP peptides causes a feedback stimulation of transcription of its gene or stabilization of its mRNA. hepatocyte growth factor receptor (Komada et al. but spermatogenesis appears normal. MAPKs have been implicated in sperm capacitation and acrosome reaction. Collectively.. ProIGFs: It is possible that implantation and placentation defects also contribute to the subfertility of PCSK4-null female mice. 2007).. they have been shown to stimulate steroidogenesis in Leydig cells (El-Gehani et al. The activating endoproteolysis could be mediated not only by PCSK4. 2000). 2000. Like PCSK4. steroidogenesis in the testes of mutant mice is lower than in those of wild. 2008)... 2006).... Their primary sequences carry several potential cleavage sites for PCSK4. 2005). 1995). The possibility of an implantation defect in PCSK4-null mice has not been explored.

An initial northern blot survey showed that most neuroendocrine cell lines contain a 1. suggesting a coordinate up-regulation PCSK activities. Alternatively. but is not restricted to. These include liver. Such injuries could be reflected by abnormal transcription of the gene. especially in neuronal and endocrine systems. in fetuses and neonates (Qiu et al. Using a PCSK4-specific fluorogenic substrate. 2008). abnormal expression or impaired activity? Could PCSK4 be a target for male contraception? PCSK4: an antisperm contraceptive target? The absence of overt spermatogenesis or steroidogenesis defects in male PCSK4-null mice suggests that therapeutic targeting of PCSK4 for contraceptive purposes in man may not be associated with the endocrinological pitfalls of hormonal contraception. 2005).6 kb PCSK4-specific transcript (Seidah et al. In the western world. Clinical implications of PCSK4 for human fertility The body of evidence discussed above strongly indicates that PCSK4 is important for male fertility in mouse. 2003). 2006). In humans. pituitary gland and various regions of the brain. 2002).PCSK4 protease and fertility 243 pregnancy. it may be worth examining whether supplementation of purified recombinant PCSK4 to deficient sperm could improve their oocyte fertilization ability in vitro. A synthetic peptide based on the sequence overlapping the demarcation between the pro and the catalytic domains of PCSK4 has recently been shown to inhibit this enzyme at low micromolar concentrations (Basak et al. Restricting this specificity remains a major challenge.. 1994). this would further support the suggestion that PCSK4 is a critical proIGF2 convertase. However. If PCSK4 transcripts can be unambiguously detected in ejaculated human sperm. a PCSK2-specific inhibitor. this attachment. Because ejaculated sperm can be obtained with relative ease. defects of placental IGF signaling have been associated with intrauterine growth restriction (IUGR) (Laviola et al. It is transmitted to the oocyte upon fertilization and may be of importance for early development. An assay for the overall PCSK4 activity per a given sperm number or per levels of immunoreactive PCSK4 may provide some indication of the fertilizing ability of the sperm pool. 2008) is a recent precedent for a non-enzymatic function of a member of the PCSK family... either because of failed expression. inactive or inhibitory. PCR-based profiling of Pcsk4 mRNA isoforms in ejaculated sperm could be used to assess the quality of PCSK4 gene expression. these isoforms could be active. 2007). Could PCSK4 deficiency contribute to some of the idiopathic infertilities. like in mouse. Half of these cases are due to the male factor infertility (MFI).. This limited transcriptome is a remnant of transcriptional events that took place during spermatogenesis (Grunewald et al. prodomain-based inhibitors often exhibit broad specificity for members of the PCSK family (Fugere et al. 2006). 2005a. Additional questions and considerations Could PCSK4 itself be a zona pellucida ligand? The binding of the PCSK9 to LDL at the surface of hepatocytes leading to post-endocytosis degradation of the receptor (Lopez. 2). Additional challenges include the mode of administration of the inhibitor and its immunogenicity.ch/) has revealed the presence of PCSK4 transcripts in several other tissues. Current efforts to develop PCSK-specific inhibitors take advantage of the properties of the prodomain as an intramolecular chaperone and inhibitor (Fugere and Day.ethz. On the other hand. 1994. Ostermeier et al.org/ by guest on November 27. PCSK4-based assays for sperm fertilizing ability A critical role of PCSK4 in human fertility would render unlikely intergenerational transmission of severely deleterious mutations in its gene. 2002. the remaining two-thirds are of unknown origin (Bhasin et al. Through the galactose binding-like fold and the RGD motif located in its P domain (see Fig. This wider distribution suggests that PCSK4 may subtly influence non-reproductive functions as well. 2002. Basak. Downloaded from http://humupd. respectively. If higher levels are observed in the mutant mice. whose ORF remains to be elucidated. A review of EST sequences deposited in DNA databases indicate that. one in six couples experiences fertility difficulties. 2005. 2012 Could PCSK4 affect other physiological functions? PCSK4 expression is preferentially expressed in. 2005. globus pallidus and cerebral cortex. if it occurs... In the context of artificial reproductive technology. The high conservation of its sequence among tetrapods. A number of reports of the last decade have shown that ejaculated human sperm contains a variety of mRNAs (Miller et al.. 2005). and thus contribute to the cascade of events leading to fertilization. alteration in expression due to developmental or environmental injuries could be a cause of infertility in human. especially the hypothalamic lateral nucleus. 2003).. might not involve cell– cell interactions since we have observed that acrosome-reacted sperm no longer carry PCSK4-specific immunoreactivity on its surface. Moreover. sperm PCSK4 could attach zona pellucida carbohydrates and oolemma integrins. adrenal cortex. the testicular abundance of its mRNA in mammals and its presence on human sperm plasma membrane overlying the acrosome (our unpublished data) suggest that it may be critical for human fertility too. The blood levels of this precursor in wildtype and PCSK4-null mice have not been compared.. reproductive tissues.. 2005).genevestigator. 2002. Topical application in the female reproductive track is a conceivable .. Miller and Ostermeier. changes in its RNA content and profile can be used to assess fertility and the consequences of environmental toxicants on fertility (Ostermeier et al. However.. 1999. human PCSK4 gene is transcribed in multiple mRNA isoforms resulting from exon skipping and optional exon insertion and encoding PCSK4 isoforms with inframe or frameshift insertions/ deletions. b. this activity could be deduced from the total PCSK-like hydrolytic minus the remaining activity in the presence of an excess of PCSK4-specific inhibitor. Miller and Ostermeier.. Brugh et al. a meta-analysis of numerous DNA microarray data (https://www. including that of PCSK4. Grunewald et al. has been used in a similar manner to measure the activity of this convertase in various mouse tissues (Li et al. their splicing profile could possibly be used as a marker of sperm quality.. One-third of MFI cases are associated with chromosomal abnormalities. Enzymatically. 2005) and IUGR has been associated with higher circulating levels of unprocessed proIGF2 (Qiu et al.oxfordjournals. 7B2.

Lazure C. Rhainds D. The role of PCSK4 in folliculogenesis. The enzymatic link could be validated by cotransduction of the putative substrate and the enzyme in an appropriate cell line. Functional processing of fertilin: evidence for a critical role of proteolysis in sperm maturation and activation. Ajoy Basak and ´ Michel Chretien for their critical reading of this manuscript before submission. Bunch DO. Benjannet S. Enzymic characterization in vitro of recombinant proprotein convertase PC4. Primakoff P. Usac EF. Creemers JW. Subtilase-like pro-protein convertases: from molecular specificity to therapeutic applications. Chen MS. Thulin CD. In vitro elucidation of substrate specificity and bioassay of proprotein convertase 4 using intramolecularly quenched fluorogenic peptides.269:9298 –9303.281:30561 –30572. The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is required for enzyme activation.271:25284 – 25291. Activation of the furin endoprotease is a multiple-step process: requirements for acidification and internal propeptide cleavage.16: 1508 – 1518. Endocrinol Metab Clin North Am 2003.343:29– 37. Analysis of mouse fertilin in wild-type and fertilin beta(2/2) sperm: evidence for C-terminal modification. Naor Z. Primakoff P. 2012 . 13:4960 – 4971. Myles DG. appropriate bioinformatic algorithms could be used to explore other biological pathways. Creemers JW. Inhibitors of proprotein convertases. Jansen E. Lipshultz LI. Rondeau N.60:117 – 126. Wickham L. Mbikay M. Branciforte D. With the availability of a mouse model of genetic PCSK4 deficiency. Mucosal or humoral antibodies induced by this application may eventually counter the contraceptive effect of the inhibitor. Chretien M. 83:844 – 855. Dev Biol 2000. Seidah NG. Mol Cell Endocrinol 2008. EMBO J 1997. Benjannet S. Gyamera-Acheampong and Mbikay Conclusion Limited endoproteolysis of precursor proteins is a mechanism evolved by all cells to regulate the production and diversity of bioactive proteins.222:289 – 295. Blobel CP. VanSlyke JK. Varret M. Seidah NG. Chretien M. sulphation and identification of the intracellular site of prosegment cleavage of PC1 and PC2. Hamelin J. Cho C. these proteins are largely unknown. 24:1 – 22. Fertilization defects in sperm from mice lacking fertilin beta. Breslin MB. Boudreault A. embryo development and implantation as well as fetal growth remains to be defined. J Mol Med 2005. Science 1998. Male factor infertility. Bergeron F. J Biol Chem 1996. Mbikay M. Rhainds D. Funding Natural Sciences and Engineering Council of Canada. J Biol Chem 1994. Basak S. Mathis JP. Matschke HM. J Biol Chem 1993. Asselin MC. J Clin Endocrinol Metab 1994. Faure JE. Differential processing of proenkephalin by prohormone convertases 1(3) and 2 and furin. Currently. Osbourne L. Bigler D. The enzyme may influence these events through its unique ability to process proIGFs to their most active forms. PCSKs are major mediators of this processing in the secretory pathway of eukaryotic cells. Cho C. Expression of the dibasic proprotein processing enzyme furin is directed by multiple promoters. Identification of these substrates is a necessary step toward elucidating the cascade of cellular events initiated by PCSK4 cleavages and leading to sperm acquisition of fertilization competence.380:505–514.32:689 – 707. J Biol Chem 2002. Bhasin S.282:39 – 44. The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications. Creemers JW. Paquet L. Success in this effort will depend on a better understanding of the biological functions of PCSK4 and its natural substrates. Shimamura S. Lazure C. These challenges could eventually be surmounted. Biochem J 1999. Bright NA. 279:48865– 48875.275:11576– 11584. Knock-out mouse models of proprotein convertases: unique functions or redundancy? Front Biosci 2008. J Biol Chem 2006. and lack of essential role of fertilin alpha in sperm-egg fusion. Downloaded from http://humupd. 79:1525 – 1529. Since there is a cornucopia of global gene expression data in health and disease online. Jin W.244 approach. Jean F. covalent post-translational modifications and efficiency of prosegment cleavage of the prohormone convertases PC1 and PC2: glycosylation. Basak A. Ge H. Van de Ven WJ. J Mol Endocrinol 2000. Roebroek AJ. The preferential expression of PCSK4 in vertebrate male gonads implies that this enzyme is particularly adapted to mediate the activation of secretory proteins of relevance for male fertility.277:12879 – 12890. Lindberg I. Thomas G. ´ Benjannet S. White JM. Khatib AM. Eddy EM. 5:75 – 83. Essalmani R. Toure BB. Clinical review 64: pathophysiology and natural history of male infertility. Rev Reprod 2000. Seidah NG.org/ by guest on November 27. this primarily gonadal protease. Molloy SS. J Biol Chem 2004. Basak A. Baker HW.oxfordjournals. de Kretser DM. Takahashi Y. Canadian Institutes of Health Research. may play more subtle physiological roles. Fei H. Jean F. Comparative biosynthesis. Mitogen activated protein kinases (MAPKs) as regulators of spermatogenesis and spermatozoa functions. Identification of a transferable sorting domain for the regulated pathway in the prohormone convertase PC2. Almeida EA. Biochem J 2004. Protein Expr Purif 2008. Mayne J. Thomas G. Acknowledgements The authors are grateful to Mrs Haidy Tadros and Francine Sirois for their technical assistance and Drs R-Marc Pelletier. Ayoubi TA. Mbikay M. Myles DG. References Almog T. Hamelin J. comparative proteomics and peptidomics of wild-type and mutant germ cells and spermatozoa could be conducted to search for these substrates. Recombinant proprotein convertase 4 (PC4) form Leishmania tarentolae expression system: purification. Leduc R. Basak A. Role of the alpha(6) integrin subunit. where PCSK4. Van de Ven WJM. Shervani N. Day R. Lazure C.294:735 – 743. Benjannet S. NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.268:27084– 27093. Nassoury N. Sequence-specific interaction between the disintegrin domain of mouse ADAM 2 (fertilin beta) and murine eggs. alpha/beta dimerization. Van de Loo JW. Brugh VM 3rd. Kolajova M.281:1857 – 1859. biochemical study and inhibitor design. Seidah NG. J Biol Chem 2000. Goulding EH. Anderson ED. Chretien M. Basak A. Anderson ED. Hutton JC. Allard D et al. Biochem J 1993.

J Biol Chem 2001. Li M. bioactive PACAP38 and PACAP27 in transfected rat pituitary GH4C1 cells. Salameh W. Tena-Sempere M. reduced binding to egg zona pellucida. Li M. Jin Y. Hobbs HH. de Potter CR. Nakayama K. Pituitary adenylate cyclase activating polypeptide-mediated intracrine signaling in the testicular germ cells. Kilpatrick DL. Analysis of O-glycosylation and endoproteolysis. Goldstein JL. Somogyvari-Vigh A. Orci L. Carroll R. Liu YJ.146:1498– 1505. Mbikay M. Biol Reprod 2000. Vilain E.110:197 – 205. Bammer S. Espenshade PJ. Tantibhedhyangkul J. Zalata A. Chretien M. Pro-protein convertases (PCs) other than PC6 are not tightly regulated for implantation in the human endometrium. Okabe M. Reproduction 2007. Gardner P. Li M. Hamelin J. Huhtaniemi I.221:66 – 72. Mol Endocrinol 2001. The insulin-like growth factors and feto-placental growth. Biol Reprod 1998. Intrauterine growth restriction in humans is associated with abnormalities in placental insulin-like growth factor signaling. Sherwood NM. Waschek JA. Mbikay M. Mol Hum Reprod 1996. Regul Pept 2003. Comhaire FH. Targeted disruption of the pituitary adenylate cyclase-activating polypeptide gene results in early postnatal death associated with dysfunction of lipid and carbohydrate metabolism. Mowen KA. precocious acrosome reaction. Isaac ER. Lavigne P. Schultz RM. PCSK9 is phosphorylated by a Golgi casein kinase-like kinase ex vivo and circulates as a phosphoprotein in humans. Pasquato A. Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins. Lewis BP. Marvaldi J. Leonardini A. J Biol Chem 1998. Day R. Naqvi S. Mature human spermatozoa do not transcribe novel RNA. Endocrinology 1998. Nature 1997. Depuydt CE.105:5750 – 5755. Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein. Downloaded from http://humupd. Fowden AL. Lindberg I. Marcinkiewicz J. Day R.59: 145 – 152. Evans JP. Post-translational processing of the insulin-like growth factor-2 precursor. Molloy SS. Perrini S.oxfordjournals. Delayed testicular aging in pituitary adenylate cyclase-activating peptide (PACAP) null mice.387:607 – 611. Steiner DF. Klarskov K. Endocrine 2004. Giorgino R et al. Cutting back on pro-protein convertases: the latest approaches to pharmacological inhibition. Duguay SJ. Lelievre V. Horton JD. Goldstein JL. Proc Natl Acad Sci USA 2003. Shioda S. SREBPs: transcriptional mediators of lipid homeostasis. The putative chaperone calmegin is required for sperm fertility. Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. Expression and localization of PCSK9 in rat hepatic cells. Karagyozov LK. Ravazzola M.280:13 – 19. Arimura A. Brown MS. FEBS J 2008.103:3793– 3798. Day R. Gray SL. Severe defect in proglucagon processing in islet A-cells of prohormone convertase 2 null mice. J Biol Chem 2002. Prat A. Cheng D.67:491– 498.74:666– 673. Arimura A. Andre F. Dabeva MD.133:1189 – 1197. Kopf GS. Glander HJ. Mol Cell Endocrinol 2008. 275:3480– 3493. Chretien M. Ikawa M. Inhibitors of the subtilase-like pro-protein convertases (SPCs). Arimura A. Brown MS. Fugere M. Selvaggi L. Petkov PM. Exp Cell Res 1995. Mbikay M. Pelletier RM. Lue YH. Shen X. Remacle-Bonnet M. Salamonsen LA. Kilpatrick LM. Ziemba AJ. Grozdanov PN. Mbikay M. Green RE. 2012 . Seidah NG. Proc Natl Acad Sci USA 2008. Gyamera-Acheampong C. Ito F. Cummings KJ. Muller JM. Beaulieu_Audy V. Trends Biochem Sci 2007. Peptides 1998. Fugere M. Arimura A. Zaid A.13:395 – 407. Freyer C. Evidence that pituitary adenylate cyclase-activating polypeptide is a potent regulator of fetal rat testicular steroidogenesis. Shuto Y. EMBO J 1995. Kominami K. Cohen JC. ´ Ftouhi N. Fry MD. Gene organization of the mouse pro-hormone and pro-protein convertase PC1. Li M. Sperm from mice genetically deficient for the PCSK4 proteinase exhibit accelerated capacitation. Montrone C. van de Loo JW. Biol Reprod 2006. Trends Pharmacol Sci 2005. Prohormone convertases 1 and 2 process ProPACAP and generate matured. Nishimune Y. Vimercati A. Proc Natl Acad Sci USA 1990.32:71 – 77.37:69– 71. Curr Pharm Des 2002. Neuroendocrinology 1999. Miyata A. Lehmann M. FEBS Lett 1993. Andrologia 2005. Ann N Y Acad Sci 2000b. Shibamoto S. Prohormone convertase PC4 processes the precursor of PACAP in the testis. In vivo functions of the proprotein convertase PC5/6 during mouse development: Gdf11 is a likely substrate.63:1482– 1489. Scioscia M. Li M. Gagnon F. Kitamura N. The receptor encoded by the human c-met oncogene is expressed in testicular tissue and on human spermatozoa. Walsh KA. Testis-specific prohormone convertase PC4 processes the precursor of pituitary adenylate cyclase-activating polypeptide (PACAP). Garrouste F. Natalicchio A. Nakayama K. Biochem Cell Biol 2006. Seidah NG. Bellan C.69:217– 226. 26:294– 301. DNA Cell Biol 1994. Proenkephalin products are stored in the sperm acrosome and may function in fertilization. Pommier G. Thomas G. Xu H. Parat F.276:27197 – 27202. Grunewald S. 2:2 – 8. Inhibitory potency and specificity of subtilase-like pro-protein convertase (SPC) prodomains. Heinlein UA. Mol Endocrinol 1998. Furuta M. Anderegg U. Lissitsky JC.15:1739– 1747. 19:259– 268. an integral transmembrane protein of the mouse acrosome. Dewpura T. Prohormone convertase 2 enzymatic activity and its regulation in neuro-endocrine cells and tissues.141:3723– 3730. Lacombe A. Gurley P. Mbikay M. Watanabe D.274:22795 – 22804. Nakayama K. Webb G. Stein J. Tanphaichitr N. Leduc R. Hatsuzawa K.org/ by guest on November 27. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Friedman TC. Day R. Thulin CD. J Biol Chem 1999. Limperis PC. Deficient processing and activity of type I insulin-like growth factor receptor in the furin-deficient LoVo-C5 cells. Toshimori K. Mbikay M. Delayed translation and posttranslational processing of cyritestin. Tadros H. Proteolytic processing of the hepatocyte growth factor/scatter factor receptor by furin.139:3763 – 3771.273:18443 – 18451. Li QL. Paasch U. Duguay AN. Jones BG. Brenner SE. Essalmani R. Mayne J. Nie G. Funahashi H. Jirik FR. Placenta 2003. Muffly KE. Somogyvari-Vigh A. Pituitary adenylate cyclase-activating polypeptide precursor is processed solely by prohormone convertase 4 in the gonads. Thomas L. Laviola L.87:9143 – 9147. Michelson RJ.23:59 – 75. Molecular biology of PCSK9: its role in LDL metabolism. 84:80– 92. Endocrinology 2005. Steiner DF.921:333– 339. Wada I. Endocrinology 2000a. Lawson G.277: 7648 – 7656. Sherwood NM. Raymond A. Weerachatyanukul W. and impaired fertilizing ability.328:25 – 29. Belsanti G.24:803 – 812. El-Gehani F. Seidah NG. Collard MW. Shuto Y. Kew D. Komada M. Linder B.14:5869– 5883. Fugere M.100:189 – 192. Pituitary adenylate cyclase-activating polypeptide (PACAP) is important for embryo implantation in mice.12:1619 – 1639. Roselli CE.8:549– 562. Arimura A. Chamberland A. Proc Natl Acad Sci USA 2006. Zhou A. Huggenvik JI.PCSK4 protease and fertility 245 Horton JD. van Emmelo J. Cold Spring Harb Symp Quant Biol 2002. Roles of the disintegrin domains of mouse fertilins alpha and beta in fertilization.

Rousselet E. Khatib AM. Benjannet S. Mayer G. Gyamera-Acheampong and Mbikay Parvinen M. Proprotein convertase genes in Xenopus development. Arch Androl 1999. 117:629– 641. Dix DJ.28:1680 – 1687. Toward using stable spermatozoal RNAs for prognostic assessment of male factor fertility. Scamuffa N. 1781:184– 191.org/ by guest on November 27. Downloaded from http://humupd.11:156 – 163. Biol Chem 2006. Leunissen JA. Proc Natl Acad Sci USA 1997.38:79 – 94.6:501 –523. Mol Endocrinol 1992. Arimura A. The activation and physiological functions of the proprotein convertases. Hakovirta H. Miller D.357:329– 342. Day R. Basak A. Naz RK. Mbikay M. Krawetz SA. Fradinger EA. Gruslin A. Bell M. Genomics 1994. Ostermeier GC. Smith P. Lonnerberg P. Proc Natl Acad Sci USA 2003. Qiu Q. Chretien M. Essays Biochem 2002. Ferguson-Smith AC. Raffin-Sanson ML.6:1559 – 1570. Biochim Biophys Acta 2008. Tang M. J Cell Biol 1997.94:6842– 6846. Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Cieplak P. Impaired fertility in mice deficient for the testicular germ-cell protease PC4. Mechanism of the facilitation of PC2 maturation by 7B2: involvement in ProPC2 transport and activation but not folding. Mbikay M. Seidah NG. Blobel CP. Collard MW. Tsang BK. Moldenhauer JS. Nassoury N. J Androl 2005b. Identification of the fourth member of the mammalian endoprotease family homologous to the yeast Kex2 protease. Seidah NG. Krawetz SA. Heinlein UA. Briggs D. Protein Sci 1997. Proprotein convertases: lessons from knockouts. Maniatis T. Chromosomal assignment of the genes for proprotein convertases PC4. Analysis of loss of adhesive function in sperm lacking cyritestin or fertilin beta. Seidah NG. Nishimura H.360:772 – 777. Prat A. Spermatozoal RNA profiles of normal fertile men. Liddington RC. Chretien M. Mayer G. Seidah NG. A suite of novel human spermatozoal RNAs. Substrate cleavage analysis of furin and related proprotein convertases: a comparative study. Coan PM. Gene 1999. Hamlington J. Soder O. 191:131– 145. PCSK9: an enigmatic protease. Cho C. reproduction and feeding. Snowden H.233:204 – 213. Goodrich RJ. Khatib AM. Zaid A.20:1954– 1963.246 Lopez D. Seidah NG.26:123 – 129. Ostermeier GC. Murakami K. Baba T.267:5897 – 5900. Prat A. Chretien M et al. Prat A.61:1445 – 1451. Shioda S. Seidah NG. Biochimie 1994. J Biol Chem 1992. Ikemizu J. Day R. Prog Nucleic Acid Res Mol Biol 1999. Stifani S. Seidah NG. Wong C.63:69– 108. Ishida N. Ostermeier GC. Mbikay M. Dix DJ. Christian JL. Remacle AG. 2012 . Qiu Q.83:1687 – 1694. Day R. Benjannet S. Miller D. Lum L.283:20897– 20906. Biochem J 1996. Goulet B. Fertil Steril 2005a. Asselin MC. Subtilases: the superfamily of subtilisin-like serine proteases.139:625 – 638. Engel W. Zhu X. Proprotein and prohormone convertases: a family of subtilases generating diverse bioactive polypeptides. Chretien M. Padman P. Neuroendocrine secretory protein 7B2: structure. Int J Biochem Cell Biol 2008. Sirois F.20:231– 237.237:385– 392. Evidence for distinct serine protease activities with a potential role in processing the sperm protein fertilin. Nakayama K. Seidah NG. 101:8204– 8208. Berg L. Krawetz SA. Tabibzadeh S.102:1104 – 11052. Mbikay M. Oh ES. Brain Res 1999. Ostermeier GC. Basak A. Pareek S. Biol Reprod 1999. Placental-specific insulin-like growth factor 2 (Igf2) regulates the diffusional exchange characteristics of the mouse placenta. Smith JW et al. Primakoff P. FASEB J 2006. Ratnikov BI. Shiryaev SA. Murphy RA. 848:45– 62. early postnatal. Diamond MP. Legradi G. Miller D. Prat A. Tsang BK. The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): liver regeneration and neuronal differentiation. Identification of insulin-like growth factor (IGF)-1 receptor in human sperm cell. Possible function of the ADAM1a/ADAM2 fertilin complex in the appearance of ADAM3 on the sperm surface. Testicular expression of PC4 in the rat: molecular diversity of a novel germ cell-specific Kex2/subtilisin-like proprotein convertase. Genomics 1995. Seidah NG. Goodrich RJ. Adams BA.418:236 – 243. cytosol and extracellular milieu. Kaipia A. Rollinson S. Oberwinkler H. Kim WS.314:951 – 960. Chen A. ´ Mbikay M. Tasic B. Hamelin J. Hosaka M. Krawetz SA. J Biol Chem 2008. Adham IM. Constancia M. Siezen RJ. Torii S. Savaria D. Diamond MP. Muller L.279:34957– 34962. Shamsadin R.76:197– 209. Localization of pituitary adenylate cyclase-activating polypeptide and its messenger ribonucleic acid in the rat testis by light and electron microscopic immunocytochemistry and in situ hybridization. Mali P. expression and functions. Miller D. Nayernia K. Sherwood NM. Nishimura H. Srinivasan A. Essalmani R. Jasmin SB. A complex population of RNAs exists in human ejaculate spermatozoa: implications for understanding molecular aspects of spermiogenesis. Gruslin A. J Biol Chem 2004. Peptides 2007. Reik W.283:2373– 2384.43:153 – 159. Jiang JY. Calvo F. and pregnant rats and persistence of circulating levels in postnatal life. Wei G. De Lamirande E. Myles DG. Dev Dyn 2005. Pasquato A. Krawetz SA.100:928– 933. Seidah NG. Biochem J 2001. Male mice deficient for germ-cell cyritestin are infertile. 387:871– 877. Poirier S. Kim E.oxfordjournals. Hamelin J. Baba T. Laliberte J. Wu S.327:625– 635. Nakaya K. Trends Mol Med 2005. Towards a better understanding of RNA carriage by ejaculate spermatozoa. Chretien M. Nakayama K. Schultz R. Chretien M. Khatri P. Muller L. Endocrinology 1994. Marcinkiewicz E. Isaac ER. Seidah NG. PC5. Precursor convertases in the secretory pathway. and PACE 4 in mouse and human. ´ Seidah NG. The controversy. Hum Reprod Update 2006. Nature 2002. The proprotein convertases and their implication in sterol and/or lipid metabolism. Clermont Y. Dev Biol 1997. Nakagawa T. Knocked down and out: PACAP in development. Ostermeier GC. potential and roles of spermatozoal RNA. Endocrinology 2007. J Biol Chem 2008.26:70 – 74. Branciforte DR. Tadros H. Chretien M. Leung WC. Lilford R. Lazure C. Its testis-specific expression. Proc Natl Acad Sci USA 2005. ´ El-Alfy M.40:1111 – 1125. Nakajo S. Chretien M. Pelto-Huikko M. Chretien M. Marcinkiewicz J. Cellular processing of the nerve growth factor precursor by the mammalian pro-protein convertases. Activation of endoproteolytic processing of insulin-like growth factor-II in fetal. Sibley CP. Lancet 2002. Lindberg I. Proc Natl Acad Sci USA 2004. 12:757 – 767.233:1038 – 1044. The family of subtilisin/kexin like pro-protein and pro-hormone convertases: divergent or shared functions. Dean W. Toppari J. Role of pro-IGF-II processing by proprotein convertase 4 in human placental development. Fowden AL.148:4803– 4811. Structure of the gene for the testis-specific proprotein convertase 4 and of its alternate messenger RNA isoforms. Hughes J. Chretien M. Nelsen S. Simpson EM. Expression of beta-nerve growth factor and its receptor in rat seminiferous epithelium: specific function at the onset of meiosis.135:818 –825. Lindberg I. Hamelin J. Ritzen EM et al. Biochem J 1997. Burton GJ. Seidah NG. Lerner CP. Alternative pre-mRNA splicing and proteome expansion in metazoans. Wickham L. Tadros H. Seidah NG. J Cell Biol 1992. The regulated cell surface zymogen activation of the proprotein convertase PC5A directs the processing of its secretory substrates. Dev Biol 2001. The cell biology of the prohormone convertases PC1 and PC2. Nakanishi T. Gaspar A.

Creemers JW.180:389 – 401. kexin-like processing endoprotease. Toure BB. CD81. Vaudry H. Crit Rev Oncog 1993. but is not essential for trans-Golgi network localization. Rochemont J. Yamagishi T. Mayne J. Myles DG. Aberrant distribution of ADAM3 in sperm from both angiotensin-converting enzyme (Ace).52:269– 324. The testicular germ-cell protease PC4 is also expressed in macrophage-like cells of the ovary. Wolfsberg TG. Seidah NG. Chretien M. Sharpe RM.272:27116 – 27123. Pituitary adenylate cyclase-activating polypeptide and its receptors: from structure to functions. Vindrola O. Zhu XR. Mbikay M. Yuan R. within mouse testis by in situ hybridization.19:1973 – 1980.137:105 – 112. Mol Endocrinol 1992. and CD98. Sakai E.4:115 –136. Lindberg I. Biosynthesis and enzymatic characterization of human SKI-1/S1P and the processing of its inhibitory prosegment. 2008 St. Young BD. Zhang CL. Disruption of PC1/3 expression in mice causes dwarfism and multiple neuroendocrine peptide processing defects. Biosynthesis. J Endocrinol 1995. Van Duijnhoven HL. Van De Ven WJ. The proprotein convertases. ADAMs in fertilization and development. Zhou A. in mouse sperm-egg plasma membrane adhesion and fusion. Van de Ven WJ. FASEB J 2003. Creemers JW. Nakayama K. J Biol Chem 2000. Structure and function of eukaryotic proprotein processing enzymes of the subtilisin family of serine proteases. Curr Opin Chem Biol 1998. Laurent V. Pituitary adenylate cyclase activating polypeptide can regulate testicular germ cell protein synthesis in vitro.144:215 – 223. Carroll R. Genomic organization and alternative splicing of human PACE4 (SPC4).and calmegin (Clgn)-deficient mice. Murakami K. Germain C. Vaudry D. 99:10293– 10298. Downloaded from http://humupd. Munzer JS.122:438 – 452. Mammalian sperm proteins are rapidly evolving: evidence of positive selection in functionally diverse genes. Pharmacol Rev 2000. Yonemoto K. Fournier A. Dey A. accepted on November 24. A role for the disintegrin domain of cyritestin.49:133 – 152. Expression and transient nuclear translocation of proprotein convertase 1 (PC1) during mouse preimplantation embryonic development. Mori K. Curbing activation: proprotein convertases in homeostasis and pathology. Lazure C.6:1088 – 1094. J Biol Chem 1997.oxfordjournals. Holst JJ et al. Biosynthesis of the prohormone convertase mPC1 in AtT-20 cells. Dev Biol 1996. Singh RS. Primakoff P. Taylor NA. Kulathinal RJ.72:483 – 493. Creemers JW.275:2349 – 2358. Localization of Kex2-like processing endoproteases.org/ by guest on November 27. Biochem J 2000. Bright NA. van de Loo JW. Dynamic palmitoylation of lymphoma proprotein convertase prolongs its half-life. White JM. Ugleholdt R. Mol Biol Cell 2001. 2:31 – 39. Tsuji A. J Biochem 1997. Proc Natl Acad Sci USA 2002. Pauli I. Sequence-specific interaction between the disintegrin domain of mouse ADAM 3 and murine eggs: role of beta1 integrin-associated proteins CD9. Basak A. distinct post-translational modifications. Takahashi Y.316:12 – 16. and functional characterization of lymphoma proprotein convertase. Tadros H. Moss SB. Torgerson DG.17:1215– 1227. Mol Biol Evol 2002. Saunders PT. Yon L.PCSK4 protease and fertility 247 van de Loo JW. Lindberg I. Steiner DF. Yoshida S. Akamatsu T. Chretien M. Bando M. furin and PC4. Biol Reprod 2006. Hine C. Bigler D. 2012 . Matsuda Y. Norrbom C. Okabe M. 75:760– 766. White JM. Van de Ven WJ. Ito Y. Torii S. Teuchert M. Roebroek AJ. J Cell Biol 1997. Ikawa M. Mol Reprod Dev 2005. Yamaguchi R. a sperm surface protein belonging to the ADAM family. J Reprod Immunol 2001. Chretien M. Submitted on July 6. Baltz JM. Plets E. 2008. Basille M.12:809 – 820. McKinnell C. Roebroek AJ.352:827 – 833. resubmitted on November 6. Benjannet S. 2008. Van de Ven WJ. Yamagata K. FEBS Lett 1993. Croissandeau G. Mbikay M. Tamai Y. Gonzalez BJ. West AP.