Identification of calcium-binding proteins associated with the human sperm plasma membrane.
BACKGROUND
The precise composition of the human sperm plasma membrane, the molecular interactions that define domain specific functions, and the regulation of membrane associated proteins during the capacitation process, still remain to be fully understood. Here, we investigated the repertoire of calcium-regulated proteins associated with the human sperm plasma membrane.


METHODS
Surface specific radioiodination was combined with two-dimensional gel electrophoresis, a 45Ca-overlay assay, computer assisted image analysis and mass spectrometry to identify calcium-binding proteins exposed on the human sperm surface.


RESULTS
Nine acidic 45Ca-binding sperm proteins were excised from stained preparative 2D gels and identified by mass spectrometry. Five of the calcium binding proteins; HSPA2 (HSP70-1), HSPA5 (Bip), HYOU1 (ORP150), serum amyloid P-component (SAP) and protein kinase C substrate 80K-H (80K-H) were found to be accessible to Iodo-Bead catalyzed 125I-labelling on the surface of intact human sperm. Agglutination and immunofluorescence analysis confirmed that SAP is situated on the plasma membrane of intact, motile sperm as well as permeabilized cells. Western blot analysis showed increased phosphorylation of human sperm 80K-H protein following in vitro capacitation. This is the first demonstration of the 80K-H protein in a mammalian sperm.


CONCLUSIONS
The presence of SAP on the surface of mature sperm implies that SAP has a physiological role in reproduction, which is thought to be in the removal of spermatozoa from the female genital tract via phagocytosis. Since 80K-H is a Ca2+-sensor recently implicated in the regulation of both inositol 1,4,5-trisphosphate receptor and transient receptor potential (TRP) cation channel activities, its detection in sperm represents the first direct signaling link between PKC and store-operated calcium channels identified in human sperm.
Naaby-Hansen
Soren
S
Department of Clinical Immunology, Aalborg Sygehus, Arhus University Hospital, Aalborg, Denmark. sonh@rn.dk
Diekman
Alan
A
Shetty
Jagathpala
J
Flickinger
Charles J
CJ
Westbrook
Anne
A
Herr
John C
JC
D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D000091342:Endoplasmic Reticulum Chaperone BiP; D006801:Humans; D008297:Male; D055101:Semen Analysis / Q000379:methods; D013073:Sperm Agglutination / Q000502:physiology; D013075:Sperm Capacitation / Q000502:physiology; D013094:Spermatozoa / Q000378:metabolism*; D055815:Young Adult
D002135:Calcium-Binding Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D002118:Calcium