Oligomerization and Transglutaminase Cross-linking of the Cystatin CRES in the Mouse Epididymal Lumen: POTENTIAL MECHANISM OF EXTRACELLULAR QUALITY CONTROL

Hans H Von Horsten,Seethal S Johnson,Susan K Sanfrancisco,Mary Catherine Hastert,Sandra M Whelly,Gail A Cornwall

doi:10.1074/jbc.m703956200

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.

Highlights

As spermatozoa migrate through the long convoluted tubule known as the epididymis, they undergo maturation and acquire motility and fertility
Cystatin C forms part of the amyloid deposits in patients with cerebral amyloid angiopathy [15], including that associated with Alzheimer disease [16], whereas the L68Q variant form of cystatin C forms amyloid deposits in the cerebral blood vessels of patients who suffer from hereditary cystatin C amyloid angiopathy and who die from hemorrhagic stroke by the age of 30 [17]
Amyloid fibrils have been found in the blood vessels of the testis and epididymis [19] and within the seminiferous tubules of the testis [20], and L68Q cystatin C amyloid deposits have been found in the testes of hereditary cystatin C amyloid angiopathy patients, suggesting the potential for reproductive pathologies as a result of amyloid formation [21]

Summary

Introduction

As spermatozoa migrate through the long convoluted tubule known as the epididymis, they undergo maturation and acquire motility and fertility. We investigated whether an active TGase in the caput epididymal luminal fluid could be responsible for the formation of the SDS-resistant high molecular mass CRES protein complexes.

Results

Conclusion