Identification of calcium-binding proteins associated with the lymphocyte plasma membrane

The Nonidet P40 insoluble fraction of lymphocyte plasma membrane contains three polypeptides of about 68,000-, 33,000- and 28,000-Mr which are solubilised by Ca2+-chelators. As judged by various criteria the 33,000-Mr polypeptide is homologous to the 36,000-Mr pp60src kinase substrate of chicken fibroblasts and the 68,000-Mr polypeptide is related to the 67,000-Mr "calelectrin" of bovine liver. The 28,000-Mr polypeptide may also be related to calelectrin.     

Identification of sperm forward motility-related proteins in human seminal plasma

Seminal plasma, an amorphous material that exists in semen, contains proteins related to sperm forward motility. Employing affinity chromatography with ConA beads and protein ultrafiltration, we isolated and concentrated proteins from heated human seminal plasma. Results of computer-assisted semen analyses (CASA) demonstrated that the forward motility index of bovine spermatozoa from the epididymal caput, incubated with proteins and theophylline, was significantly different from that of spermatozoa incubated with theophylline alone (P < 0.01). The electrophoreses revealed that the protein bands with high molecular weights in the gel of PAGE changed into low molecular weights in the gel of SDS-PAGE. Furthermore, proteins from a separated portion of the PAGE gel were still able to stimulate spermatozoa from the epididymal caput to gain forward motility. Two-dimensional (2D)-gel electrophoresis and mass spectrometry indicated that spots focused on the portion seemed, according to their amino acid sequences, to be like human alpha-1-antitrypsin and zinc-alpha-2-glycoprotein (ZAG) precursors. Western blot analysis showed the presence of these two proteins in seminal plasma. These proteins, related to the forward motility of spermatozoa in human seminal plasma, may play important roles during maturation of spermatozoa, from the epididymis through fertilization in the female reproductive tract.     

Schistosoma mansoni: characterization and identification of calcium-binding proteins associated with the apical plasma membrane and envelope.

Calcium-binding proteins (CaBPs) of Schistosoma mansoni were purified by hydrophobic affinity chromatography. Metabolically labeled CaBPs were characterized using SDS-polyacrylamide gel electrophoresis followed by fluorography. A number of CaBPs were detected in total tissue extracts, apical plasma membrane, and soluble fractions of the apical bilayer complex, ranging from 15 to 205 kDa in their molecular masses. No CaBPs were discerned in the envelope of the apical bilayer complex. Two CaBPs were positively identified as calmodulin and gelsolin via immunoblot analyses. The possible role of CaBPs in surface signal transduction mechanisms has also been briefly discussed.     

Interaction of calcium-binding proteins with calmodulin-dependent guanylate cyclase in Tetrahymena plasma membrane

Addition of bovine brain calmodulin and S-100 inhibited Tetrahymena calmodulin-induced stimulation of guanylate cyclase, but they did not affect enzymatic activity in the presence of calcium alone. Troponin C shows little effect on the cyclase activity regardless of the presence or absence of Tetrahymena calmodulin. The inhibitory effects of brain calmodulin and S-100 were overcome by the addition of Tetrahymena calmodulin, but not by calcium. Both calmodulins from Tetrahymena and bovine brain elicited stimulation of heart phosphodiesterase, while troponin C and S-100 did not affect the phosphodiesterase activity in the presence and absence of Tetrahymena calmodulin.     

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat

In the initial stage of ascidian fertilization sequential sperm–egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66kDa, 120kDa and 140kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata .     

Identification of epididymal proteins associated with hamster sperm.

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.     

Characterization of Bufo arenarum oocyte plasma membrane proteins that interact with sperm

Sperm-oocyte plasma membrane interaction is an essential step in fertilization. In amphibians, the molecules involved have not been identified. Our aim was to detect and characterize oocyte molecules with binding affinity for sperm. We isolated plasma membranes free from vitelline envelope and yolk proteins from surface-biotinylated Bufo arenarum oocytes. Using binding assays we detected a biotinylated 100 kDa plasma membrane protein that consistently bound to sperm. Chromatographic studies confirmed the 100 kDa protein and detected two additional oocyte molecules of 30 and 70 kDa with affinity for sperm. Competition studies with an integrin-interacting peptide and cross-reaction with an anti-HSP70 antibody suggested that the 100 and 70 kDa proteins are members of the integrin family and HSP70, respectively. MS/MS analysis suggested extra candidates for a role in this step of fertilization. In conclusion, we provide evidence for the involvement of several proteins, including integrins and HSP70, in B. arenarum sperm-oocyte plasma membrane interactions.     

Identification of phosphotyrosine in yeast proteins and of a protein tyrosine kinase associated with the plasma membrane

[32P]Phosphotyrosine was detected in a hydrolysate of yeast proteins after in vivo labeling with [32P]phosphoric acid. The phosphoamino acid was present in cells exponentially growing on glucose as well as in cells that had reached the stationary phase of growth. Also, a plasma membrane preparation was shown to phosphorylate casein on tyrosine residues.     

Maturing the sperm: unique mechanisms for modifying integral proteins in the sperm plasma membrane

The epididymis has been understudied, in part due to its cancer resistance and the development of effective technologies for sperm injection and in vitro fertilization. However, it is worthy of study because--absent advanced reproductive technology--its proper function is essential for conceiving children: sperm leaving the testis are immature and nonfertile. Epididymal functions can be divided into several general categories (1) concentration of sperm; (2) functional maturation; (3) storage in a quiescent state until ejaculation; (4) removal of degenerating sperm; (5) provision of appropriate conditions for survival; (6) transport by the myoid cells; (7) protection; (8) maintenance of the blood epididymal barrier. In the past decade investigators have focused on those maturational changes of the integral proteins of the sperm plasma membrane which are directly related to sperm-ova interactions. It has traditionally been thought that changes in the sperm plasma membrane proteins were limited to simple binding or removal of proteins or interactions with the proteases, glycosylases and glycotransferases present. However, the epididymis can also release secretory products in bulk through apical blebs and inject integral membrane proteins with epididymosomes which fuse with the plasma membrane. The epididymis also activates and cleaves enzymes present on the sperm surface (e.g., germ cell angiotensin converting enzyme), thus enabling them to modify proteins on the sperm membrane. Aside from the need to understand epididymal function relative to the sperm, basic science on epididymal physiology is warranted because it may help us understand the functioning of androgens, protection of tissues from oxidative damage, and resistance to cancer and benign hyperplasic growth.     

Seminal plasma proteins revert the cold-shock damage on ram sperm membrane

Ejaculated ram spermatozoa, freed from seminal plasma by a dextran/swim-up procedure and exposed to cold shock, were incubated with ram seminal plasma proteins and analyzed by fluorescence markers and scanning electron microscopy. Seminal plasma proteins bound to the sperm plasma membrane modified the functional characteristics of damaged spermatozoa, reproducing those of live cells. Scanning electron microscopy showed that the dramatic structural damage induced by cooling reverted after incubation with seminal plasma proteins. Assessment of membrane integrity by fluorescence markers also indicated a restoration of intact-membrane cells. This protein adsorption is a concentration-dependent process that induces cell surface restoration in relation to the amount of protein in the incubation medium. Fractionation of ram seminal plasma proteins by exclusion chromatography provided three fractions able to reverse the cold shock effect. Scanning electron microscopy also confirmed the high activity of one fraction, because approximately 50% of cold-shocked sperm plasma membrane surface was restored to its original appearance after incubation. Differences in composition between the three separated fractions mainly resulted from one major band of approximately 20 kDa, which must be responsible for recovering the sperm membrane permeability characteristic of a live cell.     

Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.     

ADAM7 is associated with epididymosomes and integrated into sperm plasma membrane

During epididymal transit, mammalian sperm acquire selected proteins secreted by the epididymis. We previously showed that a disintegrin and metalloprotease (ADAM) 7 is expressed specifically in the epididymis and transferred to the sperm surface during epididymal transit. Here, we show that mouse ADAM7 secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Furthermore, we found that ADAM7 can be transferred directly from epididymal vesicles to sperm and that it is an integral plasma membrane protein in sperm. Thus, our study provides new information regarding the unique mode of secretion and interaction of ADAM7 during the epididymis-to-sperm transfer process.     

Identification of synaptic plasma membrane proteins co-precipitated with fibrillar beta-amyloid peptide

The beta-amyloid peptide that is overproduced in Alzheimer's disease rapidly forms fibrils, which are able to interact with various molecular partners. This study aimed to identify abundant synaptosomal proteins binding to the fibrillar beta-amyloid (fAbeta) 1-42. Triton X-100-soluble proteins were extracted from the rat synaptic plasma membrane fraction. Interacting proteins were isolated by co-precipitation with fAbeta, or with fibrillar crystallin as a negative control. Protein identification was accomplished (1) by separating the tryptically digested peptides of the protein pellet by one-dimensional reversed-phase HPLC and analysing them using an ion-trap mass spectrometer with electrospray ionization; and (2) by subjecting the precipitated proteins to gel electrophoretic fractionation, in-gel tryptic digestion and to matrix-assisted laser desorption/ionization time-of-flight mass measurements and post-source decay analysis. Six different synaptosomal proteins co-precipitated with fAbeta were identified by both methods: vacuolar proton-pump ATP synthase, glyceraldehyde-3-phosphate dehydrogenase, synapsins I and II, beta-tubulin and 2',3'-cyclic nucleotide 3'-phosphodiesterase. Most of these proteins have already been associated with Alzheimer's disease, and the biological and pathophysiological significance of their interaction with fAbeta is discussed.     

Lipoprotein-binding proteins in the human platelet plasma membrane

The binding of homologous plasma lipoproteins to specific receptor proteins in the plasma membrane of human blood platelets was studied by ligand blotting techniques. HDL3, HDL2 and LDL showed saturable binding to three bands of 156, 130 and 115 kDa, respectively. This binding was not markedly affected by the presence or absence of Ca2+ nor by covalent modification of lysine and arginine residues of the apoprotein moieties. However, it can be almost completely reversed by the addition of heparin or suramin.     

Further characterization of boar sperm plasma membrane proteins with affinity for the porcine zona pellucida

Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.     

Characterization of the folate-binding proteins associated with the plasma membrane of rat liver

Unsaturated folate-binding proteins (i.e., apo forms) have been identified with the plasma membranes of rat liver by the binding of [3H]pteroylglutamic acid. Normal rat liver contains very little of the folate-binding apoproteins, but the folate-binding capacity increases substantially when the rats are made folate-deficient. This increase appears to be due to unsaturation of the folate-binding holoproteins rather than to synthesis of additional protein, because the binding capacity of the plasma membranes from normal rat liver following dissociation of the bound folate is equivalent to the binding capacity of the preparation from folate-deficient liver. Two molecular forms of folate-binding protein were identified by gel filtration of the solubilized plasma membrane fraction, a high-molecular-weight form (Mr less than 100,000), representing 25% of the binding capacity, and a smaller protein (Mr approximately equal to 55,000), representing 75% of the binding capacity. Whereas the larger species can be solubilized only with a detergent, the smaller form appears to be hydrophilic and dissociates spontaneously from the membrane preparation. The binding of [3H]pteroylglutamic acid by the membrane preparation was specific, saturable, and pH- and temperature-dependent. Scatchard analysis of the binding could be fitted to a curvo-linear plot, indicating at least two orders of binding sites which probably correspond to the two molecular forms identified by gel filtration. Competitive inhibition by folate analogues demonstrated that the apoproteins have higher affinity for oxidized folate than for N5-methyltetrahydrofolate and virtually no affinity for N5-formyltetrahydrofolate or methotrexate.     

Identification of ampullary and isthmic oviductal fluid proteins that associate with the bovine sperm membrane

The objective of this study was to define the complement of ampullary and isthmic oviductal fluid proteins that associate with the sperm membrane during the non-luteal and luteal stages of the oestrous cycle. Oviductal fluid was obtained from three dairy cows via indwelling cannulae in the ampulla and isthmus of the same oviduct. Daily samples of oviductal fluid were combined by region and stage of the cycle to create pools consisting of non-luteal isthmic, luteal isthmic, non-luteal ampullary, and luteal ampullary oviductal fluid. An aliquot from each oviductal fluid pool was biotinylated by incubation in biotin reagent. Spermatozoa from three fertile bulls were pooled, washed and incubated for 4 h in each of the four pools of biotinylated oviductal fluid. After incubation, sperm membrane proteins were solubilized, and proteins subjected to one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biotinylated oviductal fluid proteins that associated with the sperm membranes were detected by avidin–horseradish peroxidase and diaminobenzidine substrate. Eight oviductal fluid proteins with apparent molecular weights of 97, 75, 66, 55, 48, 34, 28 and 24 kDa were consistently detected to associate with the sperm membrane. Differences in ampullar and isthmic fluid proteins that associated with sperm during the luteal and non-luteal stage of the oestrous cycle were not detected.     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.     

Characterization of two antigenically related integral membrane proteins of the guinea pig sperm periacrosomal plasma membrane

The periacrosomal plasma membrane of mammalian spermatozoa functions both in recognition and in binding of the egg's zona pellucida and in the acrosome reaction. This study characterizes two antigenically related proteins with molecular weights of 35 kD (PM35) and 52 kD (PM52) of the guinea pig sperm periacrosomal plasma membrane. Polyclonal antisera were prepared against electrophoretically purified PM35 or PM52. Each antiserum recognized both the 35-kD and 52-kD polypeptides on Western blots, indicating that they are structurally related. This conclusion was supported by peptide mapping experiments demonstrating comparably sized fragments of both PM35 and PM52. Both PM35 and PM52 behave as integral membrane proteins during phase-separation analysis with Triton X-114. Electron microscopic immunocytochemistry and differential fractionation of sperm membranes established that both PM35 and PM52 are exclusively localized to the periacrosomal plasma membrane. Three different antisera were used for ultrastructural studies, and each specifically bound the cytoplasmic but not the extracellular membrane surface. The electrophoretic mobilities of the PM35 and PM52 polypeptides were unchanged during sperm maturation and during the ionophore-induced acrosome reaction. The localization of PM35 and PM52 suggests a potential role for these integral plasma membrane proteins in signal transduction or membrane fusion events of the acrosome reaction. © 1994 Wiley-Liss, Inc.