CABYR, a Novel Calcium-Binding Tyrosine Phosphorylation-Regulated Fibrous Sheath Protein Involved in Capacitation

Abstract

To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound (45)Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation.

Proteomic identification of human sperm proteins

“…Recently an approach to specifically extract, separate and identify human protamines by MS has been described [8]. Other proteins not identified in the present work are A kinase anchoring proteins (AKAPs), possibly because their molecular weight is out of the range studied in the present work [11]. Among nuclear proteins identified, one surprise has been the identification of RuvB-like 1 protein known to have ATPase and DNA helicase activities and to be part of the NuA4 histone acetyltransferase complex.…”

“…The general lysis and extraction procedure used has the advantage that it provides a general idea of the types of proteins that constitute the sperm proteome. Therefore, this approach should be complementary to other proteomic approaches focusing on specific compartments or organelles such as the cytoplasm, membrane, cytoskeleton, tail, acrosome or sperm head [8][9][10][11][24][25][26][27][28].…”

“…Many efforts to identify sperm proteins have been directed to the nuclear proteins [1,[5][6][7][8], to identify the proteins required for sperm motility [9][10], and in the understanding of the membrane proteins required for capacitation, egg interaction and fertilization [11]. So far, most of the fundamental knowledge on the sperm protein composition has been gained using conventional protein purification and identification strategies [1-6, 12, 13].…”

“…In man, specific approaches to target subcellular organelles or compartments have been successfully used [11,[24][25][26][27][28]. A 2-D protein map of the human sperm head has been described, but without MS identification [29].…”

Characterization of 3-hydroxyisobutyrate dehydrogenase, HIBADH, as a sperm-motility marker

“…This approach has been used specifically to discover differential sperm-related proteins (e.g., sperm-surface proteins and antisperm antibodies) [21][22][23]. In other studies, calcium-binding proteins and proteins undergoing tyrosine phosphorylation during capacitation were identified using the proteomic approach [24,25]. A modified proteomic method, difference 2D gel electrophoresis, was developed to analyze post-translational modification of proteins during sperm maturation [26].…”