FSP95, A Testis-Specific 95-Kilodalton Fibrous Sheath Antigen That Undergoes Tyrosine Phosphorylation in Capacitated Human Spermatozoa1

Abstract

Protein tyrosine phosphorylation has been associated with both capacitation and motility of mammalian sperm. During capacitation, human spermatozoa undergo tyrosine phosphorylation of a characteristic set of proteins, only one of which has thus far been cloned and localized. We report here the sequence of a fibrous sheath protein of 95 kDa (FSP95) that undergoes tyrosine phosphorylation during capacitation of human spermatozoa and has similarity to sperm A-kinase anchor proteins (AKAPs). FSP95 is both auto- and iso-antigenic in humans as it is recognized by sera containing antisperm antibodies from infertile men and women. The 853-residue protein has a calculated molecular weight of 94.6 kDa and an isoelectric point (pI) of 6.0, and it contains multiple potential phosphorylation sites for protein kinase C and casein kinase II as well as one potential tyrosine kinase phosphorylation site at amino acid 435. The sequence has amino acid homology to mouse sperm fibrous sheath AKAP82 (pro-mAKAP82, 34% identity) and to human sperm fibrous sheath AKAP82 (pro-hAKAP82, 32% identity). The gene encoding FSP95 has 5 exons separated by 4 introns and is located on chromosome 12 at locus p13.3. Northern analysis detected a single transcript of approximately 3.0 kilobases, and Northern dot blot analysis of 50 human tissues revealed FSP95 mRNA expression only in testis. By employing sperm immobilization, indirect immunofluorescence, and immunoelectron microscopy with antisera to purified recombinant FSP95, the protein was localized to the ribs of the fibrous sheath in the principal piece of the sperm tail. FSP95 is the second fibrous sheath protein to be cloned, sequenced and localized in human spermatozoa.

SAMP14, a Novel, Acrosomal Membrane-associated, Glycosylphosphatidylinositol-anchored Member of the Ly-6/Urokinase-type Plasminogen Activator Receptor Superfamily with a Role in Sperm-Egg Interaction

“…The plasmid was then transformed into the host strain E. coli BL21 DE3. The recombinant protein was affinity-purified on a Ni 2ϩ -nitrilotriacetic acid column under denaturing conditions (16). The recombinant protein was further purified on a model 491 Prep Cell (Bio-Rad) to a single band, which was verified by SDS-PAGE and used for immunizations.…”

Moving to the beat: a review of mammalian sperm motility regulation

“…The AKAPs are a family of proteins that function to tether PKA to specific subcellular regions. In the case of sperm motility, AKAPs tether PKA to the FS of the flagellum, thus restricting the scope of action of the kinase to within close proximity of motility related targets in the axoneme (Carrera et al 1994;Mei et al 1997;Miki and Eddy 1998;Mandal et al 1999;Vijayaraghavan et al 1999).…”

“…The AKAPs are now known to be major components of the FS/principal piece in a variety of mammalian species, suggesting that their role in flagellar function is evolutionarily conserved and, therefore, critical (Turner et al 1998(Turner et al , 2005Mandal et al 1999;Moss et al 1999;Jha and Shivaji 2002). Compartmentalisation of proteins to the flagellum via AKAPs is believed to ensure that the appropriate proteins find themselves in the right place at the right time to facilitate normal flagellar function (Fig.…”

FSCB, a Novel Protein Kinase A-phosphorylated Calcium-binding Protein, Is a CABYR-binding Partner Involved in Late Steps of Fibrous Sheath Biogenesis

“…A variety of factors, including Ca 2ϩ , HCO 3 Ϫ , and H 2 O 2 stimulate soluble adenylyl cyclase leading to increased cytosolic levels of cAMP (4). This increase in cAMP then activates PKA, and the consequence is a significant increase in tyrosine phosphorylation of protein substrates localized in the flagellum such as AKAP3, AKAP4, CABYR, hsp-90, ODF2, and tubulin (3,(5)(6)(7)(8)(9)(10)(11)(12).…”

“…It functions not only as an important mechanical component of flagella, but also serves as a scaffold for both glycolytic enzymes that provide energy for sperm motility and for constituents of signaling cascades involved in regulating sperm maturation, motility, capacitation, hyperactivation, and/or acrosome reaction (25). At present, several tyrosine as well as serine/threonine phosphoproteins have been reported in human (7,26), hamster (27), and mouse spermatozoa, including AKAP3, AKAP4 (7,28,29), and CABYR (8,30), which localize to the unique cytoskeletal compartment of the sperm's fibrous sheath. Identification of additional elements in the fibrous sheath "interactome" may provide further insights regarding the role of phosphorylation in regulating sperm fertilizing ability.…”