Abstract
An amyloid matrix composed of several family 2 cystatins, including the reproductive cystatin CRES, is an integral structure in the mouse epididymal lumen and has proposed functions in sperm maturation and protection. Understanding how CRES amyloid assembles in vitro may provide clues on how the epididymal amyloid matrix forms in vivo. We therefore purified full-length CRES under nondenaturing conditions and followed its aggregation from monomer to amyloid under conditions that may approximate those in the epididymal lumen. CRES transitioned into a metastable oligomer that was resistant to aggregation and only over extended time formed higher-ordered amyloids. High protein concentrations facilitated oligomer assembly and also were required to maintain the metastable state since following dilution the oligomer was no longer detected. Similar to other amyloid precursors, the formation of CRES amyloids correlated with a loss of α-helix and a gain of β-sheet content. However, CRES is unique in that its amyloids are rich in antiparallel β-sheets instead of the more common parallel β-sheets. Taken together, our studies suggest that early metastable oligomers may serve as building blocks for functional amyloid assembly and further reveal that antiparallel β-sheet-rich amyloids can be functional forms.
Highlights
We previously established that a nonpathological amyloid matrix with putative roles in sperm maturation and protection is a normal component of the mouse epididymal lumen[1]
We further showed that the amyloid forms of CRES subgroup members are present in the epididymal amyloid matrix and that all members readily formed amyloid in vitro, suggesting these structures carry out functional roles in the epididymis[2]
Using different mouse models we further demonstrated that alterations in the levels of amyloid matrix cystatins, caused either by the loss of CRES in the CRES knockout (KO) mouse or by the overexpression of a highly amyloidogenic mutant (L68Q) cystatin C led to a disrupted amyloid matrix structure and epididymal pathology[16,17]
Summary
We previously established that a nonpathological amyloid matrix with putative roles in sperm maturation and protection is a normal component of the mouse epididymal lumen[1]. In vitro cystatin C inhibited amyloid-β fibril formation[8,9,10] and in vivo cystatin C inhibited the deposition of amyloid-β in several amyloid precursor protein mouse models[11,12] implying it plays protective rather than pathological roles Together, these studies suggest the cystatin amyloids may carry out coordinated biological functions in the epididymal lumen through their organization into a common amyloid matrix. Using different mouse models we further demonstrated that alterations in the levels of amyloid matrix cystatins, caused either by the loss of CRES in the CRES knockout (KO) mouse or by the overexpression of a highly amyloidogenic mutant (L68Q) cystatin C led to a disrupted amyloid matrix structure and epididymal pathology[16,17] These conditions included a lysosomal storage-like disease and infertility[16,17]. Unlike several previously described functional and pathological amyloids, CRES amyloids were not cytotoxic to mammalian cells
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