Oligomerization and Transglutaminase Cross-linking of the Cystatin CRES in the Mouse Epididymal Lumen

Abstract

CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDS-resistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway. CRES (cystatin-related epididymal spermatogenic), a member of the cystatin superfamily of cysteine protease inhibitors, is expressed in the epididymis and spermatozoa, suggesting specialized roles in reproduction. Several cystatin family members oligomerize, including cystatin C that forms amyloid deposits associated with cerebral amyloid angiopathy. Our studies demonstrate that CRES also forms oligomers. Size exclusion chromatography revealed the presence of multiple forms of CRES in the epididymal luminal fluid, including SDS-sensitive and SDS-resistant high molecular mass complexes. In vitro experiments demonstrated that CRES is a substrate for transglutaminase and that an endogenous transglutaminase activity in the epididymal lumen catalyzed the formation of SDS-resistant CRES complexes. The use of a conformation-dependent antibody that recognizes only the oligomeric precursors to amyloid, negative stain electron microscopy, and Congo Red staining showed that CRES adopted similar oligomeric and fibrillar structures during its aggregation as other amyloidogenic proteins, suggesting that CRES has the potential to form amyloid in the epididymal lumen. The addition of transglutaminase, however, prevented the formation of CRES oligomers recognized by the conformation antibody by cross-linking CRES into an amorphous structure. We propose that transglutaminase activity in the epididymal lumen may function as a mechanism of extracellular quality control by diverting proteins such as CRES from the amyloidogenic pathway. As spermatozoa migrate through the long convoluted tubule known as the epididymis, they undergo maturation and acquire motility and fertility. Since sperm are synthetically inactive, the maturation process requires the interaction of sperm with proteins that are synthesized and secreted in a region-dependent manner by the epididymal epithelium. Following secretion, the fate of proteins in the epididymal lumen is varied. Some proteins bind to sperm and presumably affect sperm function directly, whereas others remain in the lumen throughout the length of the tubule (1Cuasnicu P.S. Cohen D.J. Ellerman D.A. Busso D. Ros Da G Morgenfeld.V. Robaire B. Hinton B.T. The Epididymis: From Molecules to Clinical Practice. 2002: 389-404Google Scholar, 2Dacheux J.L. Dacheux F. Robaire B. Hinton B.T. The Epididymis: From Molecules to Clinical Practice. 2002: 151-168Google Scholar). Other proteins are present in the epididymal lumen for only a short time, suggesting that their continued presence may be detrimental to sperm maturation and/or epididymal cell functions, and thus selective mechanisms are in place for their removal. CRES is the defining member of a reproductive subgroup of family 2 cystatins within the cystatin superfamily of cysteine protease inhibitors (MEROPS classification subfamily I25B) (3Cornwall G.A. Hsia N. Mol. Cell Endocrinol. 2003; 200: 1-8Crossref PubMed Scopus (65) Google Scholar, 4Cornwall G.A. Orgebin-Crist M.C. Hann S.R. Mol. Endocrinol. 1992; 6: 1653-1664Crossref PubMed Scopus (108) Google Scholar). CRES is synthesized and secreted into the lumen by the epithelial cells in the most proximal part of the epididymis and then abruptly disappears from the lumen a short time later (5Cornwall G.A. Hann S.R. Mol. Reprod. Dev. 1995; 41: 37-46Crossref PubMed Scopus (51) Google Scholar). In vitro CRES does not inhibit cysteine proteases but rather inhibited the serine protease prohormone convertase 2, suggesting an intracellular rather than an extracellular role for CRES (6Cornwall G.A. Cameron A. Lindberg I. Hardy D.M. Cormier N. Hsia N. Endocrinology. 2003; 144: 901-908Crossref PubMed Scopus (59) Google Scholar). Although a function of CRES within the secretory pathway of the epididymal epithelial cells would make it dispensable once it was secreted into the luminal fluid, it is not clear why or how CRES is so rapidly removed from the lumen. One possibility is that an accumulation of CRES within the epididymal lumen may be inherently harmful to epididymal cellular function and/or sperm maturation. The cystatins are one of several classes of proteins that are known to have an intrinsic propensity for cellular toxicity because of their ability to form amyloid (7Fernandez A. Berry R.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2391-2396Crossref PubMed Scopus (77) Google Scholar, 8Fernandez A. Kardos J. Scott L.R. Goto Y. Berry R.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6446-6451Crossref PubMed Scopus (71) Google Scholar, 9Fry B.G. Genome Res. 2005; 15: 403-420Crossref PubMed Scopus (344) Google Scholar). Most proteins fold into stable three-dimensional structures that allow their proper biological function. However, for a select group of proteins, if misfolding occurs, the proteins can oligomerize and form defined higher ordered structures that can lead to fibril formation and deposition of protein aggregates or amyloid that are associated with various diseases, including Alzheimer's disease, Parkinson's disease, and type II diabetes. The 20 or so proteins that are known to form amyloid seem to be unrelated, during their aggregation they oligomeric and fibrillar structures that are with such as Congo Red and as as with conformation-dependent J.L. 2003; PubMed Scopus Google Scholar, B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Although amyloid to be the form associated with disease, studies that the oligomeric precursors to the amyloid are and may the role in B. 2003; PubMed Scopus Google Scholar). Several cystatins have to and form and oligomers in I. J. PubMed Scopus Google Scholar, J. Cell PubMed Scopus Google Scholar). cystatin C forms part of the amyloid deposits in with cerebral amyloid A. S. N. S. Mol. PubMed Scopus Google including that associated with A. B. F. J. PubMed Scopus Google whereas the form of cystatin C forms amyloid deposits in the cerebral of from cystatin C amyloid and from by the of I. A. PubMed Scopus Google Scholar). is a of suggesting that the to may be a of cystatins rather than a of family and it is that CRES may also have the propensity to form amyloid S. J. PubMed Scopus Google Scholar). amyloid can form in the epididymal lumen and affect is not However, amyloid have in the of the and epididymis J. Google and within the of the D. D. Google and cystatin C amyloid deposits have in the of cystatin C amyloid suggesting the potential for reproductive as a of amyloid formation A. PubMed Scopus (77) Google Scholar). quality control mechanisms for of proteins have only and the of proteins by and by mechanisms of protein control as however, have to be 2005; 6: PubMed Scopus Google Scholar). family of members are known to protein aggregation and the formation of molecular of and proteins and thus to protein quality control are the mass mass are that form stable protein through the formation of and J. PubMed Scopus Google Scholar). The role of in the of amyloidogenic is in that studies a role for in protein thus to the disease, such as in S. B. PubMed Scopus Google Scholar, Res. PubMed Scopus Google whereas other a of by of its protein thus the of an amyloidogenic protein into the oligomeric that is and associated with J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, A. S. S. 2005; PubMed Scopus Google Scholar, S. J. 2005; PubMed Scopus Google Scholar). Although the studies suggesting that may intracellular proteins A. S. S. 2005; PubMed Scopus Google is also present and as such may similar as a of extracellular quality of the sperm maturation process that within its the extracellular in the epididymis is from other and multiple mechanisms to amyloid formation and the potential that can be associated with In CRES is present in the epididymal lumen as high molecular mass oligomeric structures and thus may be a molecular to amyloid formation and its control in the We an endogenous activity in epididymal may be in the process and thus may be one mechanism of extracellular quality and from and was from chromatography and from from and type in a with and studies in with the of for the and of of and into as in in and to allow sperm and luminal to The was for to the spermatozoa, and the was to The was luminal in a similar the luminal for exclusion chromatography was as by for to Size luminal was a exclusion with an protein chromatography with for or by with for activity was by of into One of luminal from the and epididymal for 2 in the presence of and of activity The was by and protein in for with and in a for by a the in or was during the whereas control of of by from was for the in and in the presence or of of Some also or 20 or the by the addition of to and CRES and cystatin C proteins expressed in as (5Cornwall G.A. Hann S.R. Mol. Reprod. Dev. 1995; 41: 37-46Crossref PubMed Scopus (51) Google with one mass that CRES a mass of with the of to serine by by the that during protein and to a CRES protein in as by a mass cystatin C was of an mass and not was with of CRES in for the by Some 20 or during the CRES with was during the with and the was to for 2 The by and and of CRES was to a A. D. PubMed Scopus Google Scholar). of CRES was with for 2 in the presence of as The protein was in of and The was for in the by for The was with and then in and PubMed Scopus Google Scholar). The with for and with a mass CRES was with and with a mass by the mass with mass from a of protein from the protein as as an protein the of CRES was for the The was to of mass of Da for Da for and Da for precursors a a for as with the for the during of the for the in the and by of their The for the from its the The and other in for has and has to have a II 2003; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). was as F. 1995; PubMed Scopus Google Scholar, Scholar). a was by with and and the by from the and by with a a of for The was the and a to only into the was The was to for 20 and the was The was then for 20 in to The was in the and the was to an of of 20 of in was the into the in to the from through the the the with and and by and in of a of the by and 2003; PubMed Scopus Google Scholar). The and and the was the to a of the by (5Cornwall G.A. Hann S.R. Mol. Reprod. Dev. 1995; 41: 37-46Crossref PubMed Scopus (51) Google or by F. 1995; PubMed Scopus Google Scholar, Scholar). in and in for and with CRES antibody in in (5Cornwall G.A. Hann S.R. Mol. Reprod. Dev. 1995; 41: 37-46Crossref PubMed Scopus (51) Google Scholar). The with and for 2 with a antibody a in in with by with or and to oligomeric removed from CRES and cystatin C proteins by The was by and or for in or to allow also for 2 in the presence of of and The in to and a was a the the was The was to and then was as the antibody with the that was to then and with cystatin C or amyloid and Congo Red CRES was as and for in Some for 2 with of in and The removed from various and to for the was removed with and the with for The with a electron an of or a electron an of The time of to the various amyloid structures experiments and may of protein and the presence or of CRES in the CRES oligomers from the and for of time or CRES as for amyloid by Congo Red staining a and The then in for by in Congo Red for The in by and then with G and with a with an of CRES in the propensity of cystatins to oligomerize, CRES high molecular into the epididymal lumen. We have that CRES is synthesized and secreted into the lumen by the proximal is present in the lumen of the and then abruptly disappears from the lumen of the from the epididymal was high to and the was by exclusion chromatography by and an CRES antibody F. 1995; PubMed Scopus Google Scholar, Scholar). As in the multiple forms of including SDS-sensitive and SDS-resistant high molecular the of the and forms of CRES proteins (5Cornwall G.A. Hann S.R. Mol. Reprod. Dev. 1995; 41: 37-46Crossref PubMed Scopus (51) Google in the their presence in molecular mass that in the presence of The CRES proteins in not migrate to their molecular proteins not molecular The also SDS-resistant high molecular mass CRES that the suggesting that CRES is also present in epididymal as protein complexes. CRES in or the in the exclusion the of CRES that the SDS-resistant high molecular mass in the not to during that the SDS-resistant in the of the exclusion chromatography to epididymal from CRES type and We have that the CRES and protein are not expressed in the CRES and A. The of the SDS-resistant high molecular mass CRES in the of the epididymal from the CRES that the CRES rather than a of the antibody to protein aggregates during the the of luminal was with the The addition of in the of CRES than as by The by in the by to the cross-linking in the by in the and by the to the and CRES that CRES in of of including and oligomers of the and of SDS-resistant CRES in the the of the high molecular mass SDS-resistant CRES in the epididymis, luminal from epididymal was by exclusion chromatography and and and by and by As in 2, the high molecular mass SDS-resistant forms of CRES present in the as the CRES that may higher ordered structures of The presence of high molecular mass CRES SDS-resistant and the of in epididymal that the may be to mechanisms for CRES protein is also that the of the from may be to its into the high molecular mass CRES complexes. in the have in protein aggregation because of their ability to are to cysteine proteases and to with cysteine protease inhibitors, such as an in the epididymal luminal be for the formation of the SDS-resistant high molecular mass CRES protein complexes. to activity in luminal from various of the epididymis an The activity was in the luminal from with activity in the epididymal activity was in the presence of but also be in its whereas the activity was by the addition of experiments various inhibitors, including the J. 2005; PubMed Scopus Google and the The addition of or inhibited the activity in epididymal from the with control with or the the is for its of the activity a in the epididymal Since the is not to in the luminal be as the of the activity for and A. A. 2005; PubMed Scopus Google Scholar). CRES a for a to in vitro CRES is a substrate for and and that in the formation of an studies and a CRES protein was in a of the CRES a was that prevented and a CRES protein of that in the was with and the by and The CRES in a suggesting the formation of high molecular mass CRES that to be the The addition of the prevented the formation of the CRES as by the of CRES similar to that in time the CRES protein was not The of CRES with is not to a of CRES during the of the in CRES with showed of CRES as demonstrated that most of the CRES not the The formation of CRES in the presence of that CRES is a substrate for cross-linking and and aggregation of control oligomeric and fibrillar forms of and forms of and proteins with the conformation-dependent antibody The was and then with the antibody to the and CRES and cystatin and with the conformation-dependent antibody in The and with CRES and cystatin C the protein by by by was as a negative its does not amyloid the presence of in experiments in was with in the presence or of the substrate as the then by and Although in the CRES a in of the to presumably CRES The of the into CRES was inhibited by the as as by the with the CRES suggesting that of the and that the not with not of in including and to in CRES of In the the by of with that of with in the presence of As in of CRES by and staining a of that from and of the protein CRES with by and a similar of as that from CRES Since the and the with that one in of was a for by In to the was to and the by and as for their molecular mass to by The the CRES protein was by not that that of the in CRES is as substrate by the is of that one CRES is in the one of the for cystatin of cysteine proteases I. J. 1992; PubMed Scopus Google Scholar, U. I. D. Google Scholar). the of SDS-resistant of CRES in to endogenous CRES is a substrate for luminal was with for of time, by and As in the addition of in a in CRES and an in the formation of SDS-resistant high molecular mass CRES complexes. The formation of CRES was inhibited by and the staining of the showed that the addition of not in a cross-linking of proteins in the luminal not endogenous activity in the luminal would also the formation of high molecular mass CRES luminal was in the presence of Although the formation of high molecular mass CRES was not as as with the a formation of high molecular mass CRES was The formation of the CRES was prevented by the addition of for a in the epididymal staining of the showed that not epididymal luminal proteins during the not The to CRES and the of defined molecular mass CRES in may the of epididymal and thus activity that during the of the luminal fluid, an of the the time thus its activity J. J. PubMed Google Scholar, Res. PubMed Scopus Google and/or substrate of the with the epididymal a in substrate and formation has for and J. PubMed Scopus Google Scholar). the of of proteins are to similar aggregation and into including oligomers that are to fibril formation but may the form of J.L. 2003; PubMed Scopus Google Scholar, F. F. J. N. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar, D.M. I. D.J. PubMed Scopus Google Scholar). conformation-dependent antibody in that recognizes oligomeric forms of amyloidogenic proteins of J.L. 2003; PubMed Scopus Google Scholar, A. J. PubMed Scopus Google was in a control of oligomeric and fibrillar forms of and oligomeric forms of and from The conformation-dependent antibody only the oligomeric forms of amyloidogenic proteins, the of the antibody for J.L. 2003; PubMed Scopus Google CRES into oligomers with a to the amyloidogenic proteins in the control was with that was to oligomerize for CRES by to aggregates was not recognized by the whereas the of CRES to oligomerize was by the antibody cystatin a amyloidogenic that was to also with the whereas the form not the and with the CRES and cystatin C however, of protein in the and oligomeric of the and CRES and cystatin C for the studies that a of the forms to and molecular oligomeric structures is not to be and it not A. Kardos J. Scott L.R. Goto Y. Berry R.S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6446-6451Crossref PubMed Scopus (71) Google Scholar). however, not be by the protein with the as a of to the protein not studies that cystatin is an amyloidogenic protein and can a oligomeric in its aggregation pathway. cross-linking of CRES it from oligomeric structures recognized by the the of CRES was to oligomerize in the presence of by As in and CRES not with the of the that the of the CRES to molecular a of in the also with cystatin the showed similar of CRES and cystatin C proteins in the as as in the and oligomeric demonstrated that the of proteins in the was not to a of protein during the but rather to the formation of to the that in CRES and cystatin C proteins prevented proteins from into oligomeric by aggregates in CRES adopted structures during its as to oligomerize for and removed for by negative stain electron As in during its CRES from a by an to oligomers by structures of to short to The addition of to CRES during its the formation of amorphous aggregates from structures of the amyloid aggregation that cross-linking may CRES amyloid formation The use of the conformation-dependent Congo with high to amyloid was also to CRES forms of CRES for revealed the presence of that Congo Red thus that CRES is an amyloidogenic of CRES not fibrillar structures or Congo Red not The studies demonstrate that CRES is present in the epididymal lumen as as as oligomeric several including exclusion chromatography and showed the presence of as as SDS-resistant high molecular mass CRES in the epididymal luminal Although it is that other proteins are associated with CRES and/or are present in high molecular mass the propensity for CRES to oligomerize by the amyloid pathway in vitro and ability to the formation of CRES oligomers in the presence of in of Our ability to CRES and oligomers in an epididymal by a that CRES in the epididymal lumen. The by CRES is not known and requires In studies are to CRES may undergo three-dimensional as has demonstrated for several other cystatin family including the family 2 members cystatin C A. PubMed Scopus Google and cystatin S. J. PubMed Scopus Google Scholar, A. S. J. Mol. PubMed Scopus (77) Google and the family member cystatin S. J. PubMed Scopus Google as as other amyloidogenic proteins, including protein PubMed Scopus Google Scholar). In addition to the presence of CRES in the fluid, is with are several of to that CRES has other cystatins to by The for a protein to oligomerize by can be by that the of protein Y. D. J. Mol. 2003; PubMed Scopus Google Scholar). in the cystatin C that by the to be within a and adopted the of cystatin as a was A. PubMed Scopus Google Scholar, Y. D. J. Mol. 2003; PubMed Scopus Google Scholar). The cystatin C that is in the form of amyloid in cystatin C by is also within one of of Y. D. J. Mol. 2003; PubMed Scopus Google Scholar). similar of in the CRES including to and the to that is to be part of a suggesting that CRES may also by in such as and the of CRES and the formation of oligomeric structures recognized by a conformation-dependent antibody as as the formation of Congo Red amyloid Although the for cystatin C to oligomerize in vitro than that for cystatin C also adopted the recognized by the suggesting that the mechanisms of aggregation are similar the proteins I. J. PubMed Scopus Google Scholar, A. J. PubMed Scopus Google Scholar). of cystatin C are similar to of A. J. PubMed Scopus Google Scholar). Although CRES during its in in CRES to and cross-linking to the that as a In type and cystatin C in J. Cell PubMed Scopus Google Scholar, A. F. A. PubMed Scopus Google Scholar). The CRES and cystatin C to form may in the of their is also that the may a rather than a from a to oligomeric as for cystatin C A. J. PubMed Scopus Google Scholar). of in the of SDS-resistant CRES presence of SDS-resistant high molecular mass CRES in addition to the in luminal that several mechanisms may be in the formation and/or of CRES aggregates in the epididymal lumen. Our studies that the SDS-resistant may in from cross-linking of studies demonstrate that and endogenous CRES are for and that an endogenous activity is present in the epididymal that CRES protein into stable high molecular mass complexes. have in the reproductive including epididymis, and spermatozoa, and have to a role in of B. Endocrinology. PubMed Scopus Google formation of the A. A. J. J. PubMed Scopus Google sperm motility Res. PubMed Scopus Google and sperm PubMed Scopus Google Scholar, A. A. S. Reprod. PubMed Scopus Google Scholar). is known the or function of in the epididymis other than that sperm and from the of the epididymis higher of activity than the S. J. Google Scholar). Our studies that the activity present in the epididymal luminal is a as by of its activity in the presence of a Our studies also that the activity was in the luminal from and with the of luminal CRES a role for in cross-linking In in vitro studies that activity is J. 2005; PubMed Scopus Google studies to activity in the epididymis luminal in the epididymis, and activity was studies a within the epididymal lumen. for a role of in the formation of stable CRES protein is by the that are to cysteine and thus their is to cysteine protease inhibitors, such as the of by and that one of the was in an in cystatins and for with cysteine The cystatins and cystatin have also to be transglutaminase in vitro N. 1992; PubMed Scopus Google Scholar, F. J. J. PubMed Scopus Google Scholar). Our studies that cystatin C is also a of CRES in the studies that the and formation of high molecular mass CRES protein to the of CRES from the epididymal lumen. The that is why is CRES so rapidly that epididymal secretory proteins remain in the epididymal lumen their studies and of it is that to CRES it amyloidogenic and the proper is within the epididymal lumen in of the from the is removed by by the in a of proteins within the lumen. is to protein aggregation J. Mol. PubMed Scopus Google and thus CRES as to in the lumen. Our studies that cross-linking of CRES protein may also in the epididymal lumen. Although studies are to in CRES cross-linking or it CRES the presence of CRES in the lumen would in of the The biological role of cross-linking of CRES to be is to however, that may function as part of a quality control in the epididymal lumen. The of CRES to in vitro into oligomeric and fibrillar structures to other amyloidogenic proteins, such as and that a similar in The oligomeric forms of amyloidogenic proteins have to be and may in the form of the amyloid rather than J.L. 2003; PubMed Scopus Google Scholar). Although the formation of oligomers is a in extracellular it is in the epididymal lumen in sperm be and maturation to if of the is to the epididymis with may be to not allow CRES aggregation to to the formation of a ordered but rather in its process and form stable of oligomeric in and amorphous protein aggregates then be for in epididymal with the of the presence of the protein and in the and epididymal lumen that such a mechanism may be in place B. Reprod. 2005; PubMed Scopus Google Scholar). Although the of propose that structures may be a to proteins for maturation to the sperm the possibility of a role for CRES it is also that they proteins for studies demonstrate that of protein aggregation in the epididymis is and may of extracellular quality control that have and amyloidogenic We for with the studies and of of for of the with