Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Seven monoclonal antibodies (MAs) against 55 kDa glycoprotein family of porcine zona pellucida (ZP3) reacting with either ZP3 alpha (MA-7, MA-27, MA-28) or ZP3 beta (MA-1, MA-2, MA-11, MA-30) have been described. MA-1, -2, -27, -28 and -30 do not recognize carbohydrate determinants as shown by their reactivity to the deglycosylated (DG) ZP3 alpha and ZP3 beta. Indirect immunoperoxidase studies showed that all MAs reacted with zona pellucida from porcine and monkey ovaries. Only MA-1 and -27 reacted with ZP from rabbit ovary as well, while none of the MAs recognised mouse ZP, MA-7, -11, -27, -28 and -30 inhibited in vitro, the zona lysis by trypsin as well as the binding of ZP3 to sperm membrane vesicle as investigated by ELISA.
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PMID:Characteristics of monoclonal antibodies against porcine zona pellucida-3 and their functional relevance. 129 21

The distribution of O-linked oligosaccharides on the M(r) 55,000 glycoproteins, ZP3 alpha and ZP3 beta, of the porcine oocyte zona pellucida was examined. Purified preparations of endo-beta-galactosidase digested ZP3 alpha and ZP3 beta were reduced and carboxamidomethylated and digested with trypsin. When the trypsin digests were mapped by HPLC, each glycoprotein yielded only one N-acetylgalactosamine containing glycopeptide. Purification of the O-glycopeptides was achieved by a two-step protocol. Tryptic digests were applied to jacalin-agarose and specifically-bound O-glycopeptides (alpha OGP and beta OGP) were eluted with buffer containing 50 mM alpha-methylgalactoside as the haptenic sugar. Further purification of each O-glycopeptide was accomplished by reverse phase HPLC. Purified O-glycopeptides were characterized with respect to amino acid and carbohydrate compositions and sequenced by automated Edman degradation; alpha OGP was a 41-residue glycopeptide with three O-linked sugar chains. Sequence comparisons revealed a 75% identity between alpha OGP and a corresponding segment of rabbit rec55 zona protein; beta OGP was a 25-residue glycopeptide characterized by the presence of one N-linked and five O-linked sugar chains and a trypsin-resistant internal arginine residue. Sequence alignments revealed an 80% or greater identity between beta OGP and internal peptides of mouse, hamster and human ZP3 zona proteins. These studies demonstrate that in the case of ZP3 alpha and ZP3 beta, the pig homologues of rabbit rec55 and mouse ZP3, respectively, O-linked oligosaccharides are confined within delimited domains rather than widely dispersed on the polypeptide backbone. Such clustering of O-linked oligosaccharides may represent an essential determinant of the structure and biological activity of zona proteins.
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PMID:Porcine oocyte zona pellucida M(r) 55,000 glycoproteins: identification of O-glycosylated domains. 141 87

To study zona pellucida antigens involved in human fertilization, five monoclonal antibodies (MAbs)--2A1, 2G3, 4A2, 4E12, and 5H4--were produced to a glycoprotein family (ZP4) isolated from heat-solubilized porcine zonae pellucidae. Each MAb reacted not only with solubilized porcine zona glycoproteins but also with the glycoproteins deglycosylated by trifluoromethanesulfonic acid treatment. They also reacted with intact zonae pellucidae of porcine and human oocytes. Three (4A2, 4E12, and 5H4) of the five MAbs showed a significant blocking effect on human sperm binding and penetration of human zonae pellucidae. The 5H4 MAb showed a strong reaction with ZP4 and ZP1 glycoprotein families of porcine zonae pellucidae, and four other MAbs reacted more strongly with ZP3 than with ZP4. The reactivity of 5H4 with porcine zona glycoproteins was destroyed by chymotrypsin digestion, but the antigen epitope was resistant to proteolysis by trypsin and endoproteinase Lys-C. A peptide fragment reactive to 5H4 was isolated by reverse-phase HPLC from endoproteinase Lys-C-treated ZP4 glycoproteins, and its molecular mass was determined to be 7 kDa by SDS-PAGE. These results suggested that the antigen epitope corresponding to 5H4 is a good candidate for development of a contraceptive vaccine.
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PMID:Blocking of human sperm-zona interaction by monoclonal antibodies to a glycoprotein family (ZP4) of porcine zona pellucida. 175 10

During fertilization in mice, acrosome-intact sperm bind via plasma membrane overlying their head to a glycoprotein, called ZP3, present in the egg extracellular coat or zona pellucida. Bound sperm then undergo the acrosome reaction, which results in exposure of inner acrosomal membrane, penetrate through the zona pellucida, and fuse with egg plasma membrane. Thus, in the normal course of events, acrosome-reacted sperm must remain bound to eggs, despite loss of plasma membrane from the anterior region of the head and exposure of inner acrosomal membrane. Here, we examined maintenance of binding of sperm to the zona pellucida following the acrosome reaction. We found that polyclonal antisera and monoclonal antibodies directed against ZP2, another zona pellucida glycoprotein, did not affect initial binding of sperm to eggs, but inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. On the other hand, polyclonal antisera and monoclonal antibodies directed against ZP3 did not affect either initial binding of acrosome-intact sperm to eggs or maintenance of binding following the acrosome reaction. We also found that soybean trypsin inhibitor, a protein reported to prevent binding of mouse sperm to eggs, did not affect initial binding of sperm to eggs, but, like antibodies directed against ZP2, inhibited maintenance of binding of sperm that had undergone the acrosome reaction on the zona pellucida. These and other observations suggest that ZP2 serves as a secondary receptor for sperm during the fertilization process in mice and that maintenance of binding of acrosome-reacted sperm to eggs may involve a sperm, trypsin-like proteinase.
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PMID:Identification of a secondary sperm receptor in the mouse egg zona pellucida: role in maintenance of binding of acrosome-reacted sperm to eggs. 339 65

Out of 11 monoclonal antibodies(MAbs) developed against porcine zona pellucida-3 beta(ZP3 beta) glycoprotein, 6 (MA-451, -454, -455, -462, -467 and -470) reacted with ZP2 beta, deglycosylated ZP3 beta(DGZP3 beta), and reduced and carboxyamidomethylated ZP3 beta(RCMZP3 beta) in ELISA and Western blot suggesting that these monoclonals recognise a linear protein epitope. Five MAbs(MA-452, -456, -459, -474 and -476) showed reduced binding with DGZP3 beta and RCMZP3 beta as compared to ZP3 beta in ELISA suggesting that these may recognize either carbohydrate moities or conformation dependent epitopes. Moreover, these MAbs failed to recognize these antigens on Western blot. Based on competitive inhibition studies in ELISA for the ability of MAbs to inhibit the binding of biotinylated ZP3 beta to solid phase antibody, 6 distinct domains were discernible. Domain V recognised by MA-467 partially overlaps with the domain corresponding to MA-454 and MA-455. All 11 MAbs reacted with zona pellucida of intact oocytes as revealed by indirect immunofluorescence but only 3 antibodies (MA-454, -455 and -467) delayed the zona lysis by trypsin. In immunoblots MA-467 recognised 18kDa fragment of RCMZP3 beta digested with endoproteinase lys-C and 37 and 30kDa bands of elastase digest of ZP3 beta. The results will help in delineation of functionally relevant domains and design of contraceptive vaccine based on zona pellucida aiming for pre-fertilization block.
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PMID:Characterization of antigenic determinants on porcine zona pellucida-3 beta(ZP3 beta) glycoprotein by monoclonal antibodies. 752 69

Seven monoclonal antibodies (MAs) generated against porcine zona pellucida glycoprotein, ZP3 (comprising both ZP3 alpha and ZP3 beta) were characterized for their specificities to ZP3 alpha (MA 7 and MA 28) or ZP3 beta (MA 1, MA 2, MA 10, MA 27 and MA 30) and their relative affinities in competitive ELISA. Among the seven MAs tested, MA 28 showed the highest affinity for ZP3 and ZP3 alpha and MA 30 for ZP3 beta. All the antibodies bound to the zona pellucida in an indirect immunofluorescence assay, but only four (MA 7, MA 28, MA 10 and MA 30) were able to inhibit the binding of boar sperm to the porcine oocyte. Reduction followed by carboxyamidomethylation of the antigen or its chemical deglycosylation reduces reactivity to MA 7 and MA 10, suggesting that these antibodies read conformational or discontinuous determinants. The epitope recognized by MA 28 is sequential or conformational, stabilized by disulfide bonds while MA 30 reads a sequential determinant. ZP3 alpha digested with alpha-chymotrypsin, trypsin and V8 protease, respectively, revealed fragments in the range of 27-20 kDa with MA 28 in immunoblots. Proteolytic digests of ZP3 beta show that MA 30 recognizes approximately 14 kDa fragment of an alpha-chymotrypsin digest and a approximately 6 kDa fragment of a tryptic digest. These studies will help in delineation of smaller determinants of ZP involved in sperm binding.
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PMID:Delineation of epitopes on porcine zona pellucida relevant for binding of sperm to oocyte using monoclonal antibodies. 768 10

All 10 monoclonal antibodies generated reacted with deglycosylated ZP3 alpha both in ELISA and Western blots, thereby suggesting that these do not recognize carbohydrate determinants. Moreover, 7 antibodies (MAs -402, -403, -405, -412, -420, -421 and -423) recognized reduced and carboxyamidomethylated ZP3 alpha in Western blot suggesting that these antibodies recognized linear epitopes. The epitopes recognized by MAs -410, -413 and -425 were sequential or conformational, stabilized by disulphide bonds. Three antibodies namely, MA -405, -420 and -421 inhibited in vitro, zona lysis by trypsin.
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PMID:Generation and characterization of monoclonal antibodies to porcine zona pellucida-3 alpha glycoprotein. 822 12

Zona pellucida glycoproteins play an important role in fertilization. In this study, attempts have been made to identify and define epitopes of monoclonal antibodies (mAbs) possessing contraceptive efficacy in vitro. The porcine zona glycoprotein pZPC, a homologue of mouse/human ZP3, was reduced and alkylated and subsequently digested with trypsin. Reverse-phase HPLC of the tryptic digest yielded twenty two peaks (T1-T22). When tested against mAbs reactive against sequential determinants on pZPC, T11 was immunoreactive with two mAbs, mAb-455 and mAb-467, as shown by antigen inhibition ELISA. IC50 values of 3.1 nM and 8.6 nM were recorded versus mAb-455 and mAb-467 respectively, and approximated the IC50 values obtained with intact pZPC. Amino acid analysis, Edman degradation, and FAB-MS identified T11 as the N-blocked decapeptide pyro-Gln-Pro-Val-Trp-Gln-Asp-Glu-Gly-Gln-Arg derived from the N-terminus of pZPC. Synthesis of overlapping octapeptides further identified VWQDE and WQDE as the minimum motifs with antigenic activity for mAb-455 and mAb-467, respectively. Glycine replacement peptides confirmed residues W,Q,E as critical for binding mAb-455 and W,Q,D,E as critical for binding mAb-467. Both mAbs inhibited binding of boar sperm to zona-encased porcine oocytes. These results, the first to define peptide epitopes of porcine zona glycoprotein, will assist in the design of an immunocontraceptive vaccine based on synthetic peptides corresponding to pZPC or its homologues in other species.
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PMID:Localization of epitopes for monoclonal antibodies at the N-terminus of the porcine zona pellucida glycoprotein pZPC. 856 67

Mammalian eggs are surrounded by two egg coats: the cumulus oophorus and the zona pellucida, which is an extracellular matrix composed of sulfated glycoproteins. The first association of the spermatozoon with the zona pellucida occurs between the zona glycoprotein, ZP3 and sperm receptors, located at the sperm plasma membrane, such as the 95 kDa tyrosine kinase-protein. This association induces the acrosome reaction and exposes the proacrosin/acrosin system. Proacrosin transforms itself, by autoactivation, into the proteolytical active form: acrosin. This is a serine protease that has been shown to be involved in secondary binding of spermatozoa to the zona pellucida and in the penetration of mammalian spermatozoa through it. The zona pellucida is a specific and natural substrate for acrosin and its hydrolysis and fertilization can be inhibited by antiacrosin monoclonal antibodies. Moreover, in in vitro fertilization experiments, trypsin inhibitors significantly inhibits fertilization. The use of the silver-enhanced immunogold technique has allowed immunolocalization of the proacrosin/acrosin system in spermatozoa after the occurrence of the acrosome reaction. This system remains associated to the surface of the inner acrosomal membrane for several hours in human, rabbit and guinea-pig spermatozoa while in the hamster it is rapidly lost. In the hamster, the loss of acrosin parallels the capability of the sperm to cross the zona pellucida. Rabbit perivitelline spermatozoa can fertilize freshly ovulated rabbit eggs and retain acrosin in the equatorial and postacrosomal region. These spermatozoa also show digestion halos on gelatin plates that can be inhibited by trypsin inhibitors. This evidence strongly suggests the involvement of acrosin in sperm penetration through the mammalian zona. Recently it was shown, however, that acrosin would not be essential for fertilization. It is likely, then, that such an important phenomenon in the mammalian reproductive cycle would be ensured though several alternative mechanisms.
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PMID:Early steps of sperm-egg interactions during mammalian fertilization. 893 5

Guanidinobenzoatase (GB), a proteolytic enzyme found in the epididymal fluids of mice, was purified to apparent homogeneity by molecular sieving and affinity chromatography. It has a molecular mass of 71 kDa and its enzymatic activity is heat labile and sensitive to EGTA. Its kinetic parameters (K(m) of 6.66 microM and a Vmax of 4.38 nmol/min/mg) were determined using 4-methylumbelliferyl-p-guanidinobenzoate (MUGB) as the substrate. GB activity is concentrated in the cauda epididymal region of the genital tract. Heat-solubilized whole zonae, biologically active ZP3, and several serine proteinase inhibitors, including a proteinase inhibitor endogenous to the male genital tract, effectively block the ability of GB to hydrolyze MUGB. Pretreating cumulus-free, zonae intact oocytes with purified GB reduces, in a concentration-dependent manner, the number of sperm able to bind to the zonae. The function of the soluble enzyme is not known. Its ability to bind both trypsin inhibitors and ZP3 suggests a possible role in gamete recognition.
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PMID:Characterization of the guanidinobenzoatase in the epididymal fluids of the mouse. 913 23


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