Interactions between mouse ZP2 glycoprotein and proacrosin; a mechanism for secondary binding of sperm to the zona pellucida during fertilization

2001 ◽  
Vol 114 (22) ◽  
pp. 4127-4136
Author(s):  
Elizabeth Howes ◽  
John C. Pascall ◽  
Wolfgang Engel ◽  
Roy Jones
Keyword(s):  
Zona Pellucida ◽  
Solid Phase ◽  
Binding Assays ◽  
Vitro Fertilization ◽  
Sperm Binding ◽  
Secondary Receptor ◽  
Specificity Of Binding ◽  
Solid Phase Binding ◽  
Complementary Binding

The mouse zona pellucida glycoprotein, mZP2, is thought to be the secondary receptor on eggs for retention of acrosome-reacted sperm during fertilization. Here, we present evidence that one of its complementary binding proteins on sperm is proacrosin/acrosin. mZP2 binds to proacrosin null sperm considerably less effectively than to wild-type sperm. Binding is mediated by a strong ionic interaction between polysulphate groups on mZP2 and basic residues on an internal proacrosin peptide. The stereochemistry of both sulphate groups and basic amino acids determines the specificity of binding. Structurally relevant sulphated polymers and suramin, a polysulphonated anticancer drug, compete with mZP2 for complementary binding sites on proacrosin/acrosin in solid-phase binding assays. The same competitors also displace attached sperm from the zona pellucida of eggs in an in vitro fertilization system. This combination of genetic, biochemical and functional data supports the hypothesis that mZP2-proacrosin interactions are important for retention of acrosome-reacted sperm on the egg surface during fertilization. Safe mimetics of suramin have potential as non-steroidal antifertility agents.

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

Ability of PknA, a mycobacterial eukaryotic-type serine/threonine kinase, to transphosphorylate MurD, a ligase involved in the process of peptidoglycan biosynthesis

2008 ◽  
Vol 415 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Meghna Thakur ◽  
Pradip K. Chakraborti
Keyword(s):  
Protein Kinases ◽  
Solid Phase ◽  
Morphological Changes ◽  
Binding Assays ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Peptidoglycan Biosynthesis ◽  
Vitro Binding ◽  
Solid Phase Binding

Eukaryotic-type serine/threonine protein kinases in bacteria have been implicated in controlling a host of cellular activities. PknA is one of eleven such protein kinases from Mycobacterium tuberculosis which regulates morphological changes associated with cell division. In the present study we provide the evidence for the ability of PknA to transphosphorylate mMurD (mycobacterial UDP-N-acetylmuramoyl-L-alanine:D-glutamate-ligase), the enzyme involved in peptidoglycan biosynthesis. Its co-expression in Escherichia coli along with PknA resulted in phosphorylation of mMurD. Consistent with these observations, results of the solid-phase binding assays revealed a high-affinity in vitro binding between the two proteins. Furthermore, overexpression of m-murD in Mycobacterium smegmatis yielded a phosphorylated protein. The results of the present study therefore point towards the possibility of mMurD being a substrate of PknA.

New insights into the structural elements involved in the skin haemorrhage induced by snake venom metalloproteinases

2010 ◽  
Vol 104 (09) ◽  
pp. 485-497 ◽  
Author(s):  
Ana Oliveira ◽  
Adriana Paes Leme ◽  
Amanda Asega ◽  
Antonio Camargo ◽  
Jay Fox ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Snake Venom ◽  
Solid Phase ◽  
Structural Elements ◽  
Binding Assays ◽  
Domain Organisation ◽  
Solid Phase Binding ◽  
A Minor ◽  
Von Willebrand

SummaryHaemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon resulting in capillary disruption and extravasation. This study analysed structural elements important for the interaction of four Bothrops jararaca SVMPs of different domain organisation and glycosylation levels with plasma and extracellular matrix proteins: HF3 (P-III class) is highly glycosylated and ~80 times more haemorrhagic than bothropasin (P-III class), which has a minor carbohydrate moiety; BJ-PI (P-I class) is not haemorrhagic and the DC protein is composed of disintegrin-like/cysteine-rich domains of bothropasin. HF3, bothropasin and BJ-PI showed different degradation profiles of fibrinogen, fibronectin, vitronectin, von Willebrand factor, collagens IV and VI, laminin and Matrigel™; however, only bothropasin degraded collagen I. In solid-phase binding assays HF3 and bothropasin interacted with fibrinogen, fibronectin, laminin, collagens I and VI; the DC protein bound only to collagens I and VI; however, no binding of BJ-PI to these proteins was detected. N-deglycosylation caused loss of structural stability of bothropasin and BJ-PI but HF3 remained intact, although its haemorrhagic and fibrinogenolytic activities were partially impaired. Nevertheless, N-deglycosylated HF3 bound with higher affinity to collagens I and VI, although its proteolytic activity upon these collagens was not enhanced. This study demonstrates that features of carbohydrate moieties of haemorrhagic SVMPs may play a role in their interaction with substrates of the extracellular matrix, and the ability of SVMPs to degrade proteins in vitro does not correlate to their ability to cause haemorrhage, suggesting that novel, systemic approaches are necessary for understanding the mechanism of haemorrhage generation by SVMPs.

scholarly journals A disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif 9 (ADAMTS9) regulates fibronectin fibrillogenesis and turnover

2019 ◽  
Vol 294 (25) ◽  
pp. 9924-9936 ◽  
Author(s):  
Lauren W. Wang ◽  
Sumeda Nandadasa ◽  
Douglas S. Annis ◽  
Joanne Dubail ◽  
Deane F. Mosher ◽  
...  
Keyword(s):  
Solid Phase ◽  
Binding Assays ◽  
Dual Roles ◽  
Cultured Fibroblasts ◽  
Ecm Remodeling ◽  
Its Gene ◽  
Fibrillin 1 ◽  
Solid Phase Binding

The secreted metalloprotease ADAMTS9 has dual roles in extracellular matrix (ECM) turnover and biogenesis of the primary cilium during mouse embryogenesis. Its gene locus is associated with several human traits and disorders, but ADAMTS9 has few known interacting partners or confirmed substrates. Here, using a yeast two-hybrid screen for proteins interacting with its C-terminal Gon1 domain, we identified three putative ADAMTS9-binding regions in the ECM glycoprotein fibronectin. Using solid-phase binding assays and surface plasmon resonance experiments with purified proteins, we demonstrate that ADAMTS9 and fibronectin interact. ADAMTS9 constructs, including those lacking Gon1, co-localized with fibronectin fibrils formed by cultured fibroblasts lacking fibrillin-1, which co-localizes with fibronectin and binds several ADAMTSs. We observed no fibrillar ADAMTS9 staining after blockade of fibroblast fibronectin fibrillogenesis with a peptide based on the functional upstream domain of a Staphylococcus aureus adhesin. These findings indicate that ADAMTS9 binds fibronectin dimers and fibrils directly through multiple sites in both molecules. Proteolytically active ADAMTS9, but not a catalytically inactive variant, disrupted fibronectin fibril networks formed by fibroblasts in vitro, and ADAMTS9-deficient RPE1 cells assembled a robust fibronectin fibril network, unlike WT cells. Targeted LC-MS analysis of fibronectin digested by ADAMTS9-expressing cells identified a semitryptic peptide arising from cleavage at Gly2196–Leu2197. We noted that this scissile bond is in the linker between fibronectin modules III17 and I10, a region targeted also by other proteases. These findings, along with stronger fibronectin staining previously observed in Adamts9 mutant embryos, suggest that ADAMTS9 contributes to fibronectin turnover during ECM remodeling.

scholarly journals Mice expressing aberrant sperm-specific protein PMIS2 produce normal-looking but fertilization-incompetent spermatozoa

2012 ◽  
Vol 23 (14) ◽  
pp. 2671-2679 ◽  
Author(s):  
Ryo Yamaguchi ◽  
Yoshitaka Fujihara ◽  
Masahito Ikawa ◽  
Masaru Okabe
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Specific Protein ◽  
Aberrant Expression ◽  
Sperm Transport ◽  
Vitro Fertilization ◽  
Sperm Binding ◽  
Sperm Membrane ◽  
Domain 3

Eight kinds of gene-disrupted mice (Clgn, Calr3, Pdilt, Tpst2, Ace, Adam1a, Adam2, and Adam3) show impaired sperm transition into the oviducts and defective sperm binding to the zona pellucida. All of these knockout strains are reported to lack or show aberrant expression of a disintegrin and metallopeptidase domain 3 (ADAM3) on the sperm membrane. We performed proteomic analyses of the proteins of these infertile spermatozoa to clarify whether the abnormal function is caused exclusively by a deficiency in ADAM3 expression. Two proteins, named PMIS1 and PMIS2, were missing in spermatozoa from Clgn-disrupted mice. To study their roles, we generated two gene-disrupted mouse lines. Pmis1-knockout mice were fertile, but Pmis2-knockout males were sterile because of a failure of sperm transport into the oviducts. Pmis2-deficient spermatozoa also failed to bind to the zona pellucida. However, they showed normal fertilizing ability when eggs surrounded with cumulus cells were used for in vitro fertilization. Further analysis revealed that these spermatozoa lacked the ADAM3 protein, but the amount of PMIS2 was also severely reduced in Adam3-deficient spermatozoa. These results suggest that PMIS2 might function both as the ultimate factor regulating sperm transport into the oviducts and in modulating sperm–zona binding.

294 THE EFFECT OF PLASMIN ON THE IN VITRO FERTILIZATION ABILITY OF PORCINE GAMETES

2006 ◽  
Vol 18 (2) ◽  
pp. 254
Author(s):  
H.-H. Rhee ◽  
S.-J. Sa ◽  
H.-T. Cheong ◽  
B.-K. Yang ◽  
C.-K. Park
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Proteolytic Enzymes ◽  
Extracellular Proteins ◽  
Control Group ◽  
Sperm Viability ◽  
Vitro Fertilization ◽  
Sperm Binding

Plasminogen activators (PAs) are specific proteolytic enzymes that convert the inactive proenzyme plasminogen to plasmin. The plasmin formed is a nonspecific, potent protease that cleaves blood fibrin clots and several other extracellular proteins. The purposes of the present study were (1) to assess the effect of plamin on sperm viability and acrosome reaction (AR), (2) to examine the effect of plasmin on zona pellucida (ZP) solubility and the binding of sperm to ZP, and (3) to evaluate the effect of plasmin on fertilization responses, including penetration and incidence of polyspermy during in vitro fertilization in the pig. Ejaculated semen was collected from three mature Duroc boars by artificial vagina. The same three boars were used for all experiments. The oocyte maturation medium used was North Carolina State University-23 (NCSU-23) medium supplemented with 10% (v/v) porcine follicular fluid (pFF), 0.6 mM cysteine, 10 IU/mL human chorionic gonadotropin (hCG; Sigma-Aldrich Corporation, St. Louis, MO, USA), and 10 IU/mL pregnant mare's serum gonadotropin (PMSG; Sigma). Porcine spermatozoa, which were washed in Dulbecco PBS (Sigma), were resuspended and incubated in fertilization medium (mTBM) containing 0, 0.1, 1.0, 10.0, or 100.0 ng/mL plasmin (Sigma). Data were analyzed by ANOVA and Duncan's multiple-range test using the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The present study suggests that sperm viability was not affected by plasmin treatment. Also, addition of plasmin in doses ranging between 0.1 and 100.0 ng/mL for 2, 4, or 6 h to washed boar spermatozoa resulted in enhancement of acrosome reaction (AR), compared with untreated cells. Concentrations of 0 and 0.1 ng/mL plasmin (83 � 15 and 95 � 18 sperm/oocyte, respectively) had no effect on sperm binding, whereas 1.0 (123 � 21 sperm/oocyte), 10.0 (124 � 16 sperm/oocyte), and 100 ng/mL (124 � 15 sperm/oocyte) plasmin increased (P < 0.05) sperm binding, compared with the control. The zona pellucida solubility (zona digestion time) was significantly (P < 0.05) lower in medium with 1.0 (123 � 24 s), 10.0 (99 � 15 s), or 100.0 ng/mL (95 � 19 s) plasmin, compared with control (176 � 27 s). When porcine oocytes and spermatozoa were co-incubated in various concentrations of plasmin for 6 h, the penetration rate was significantly (P < 0.05) higher in medium with 1.0 ng/mL plasmin (77.5 � 3.1%), compared with control. However, there were no significant differences in the polyspermic rates and mean numbers of sperm (MNS)/oocyte among the groups treated with plasmin and the control group. We found that addition of plasmin to fertilization medium increases the percentage of acrosome-reacted spermatozoa and the sperm-binding ability of the pig ZP. These results suggest that plasmin may play a role in events related to fertilization in the pig.

scholarly journals Sperm surface galactosyltransferase activities during in vitro capacitation

1982 ◽  
Vol 95 (2) ◽  
pp. 567-573 ◽  
Author(s):  
BD Shur ◽  
NG Hall
Keyword(s):  
Molecular Weight ◽  
Zona Pellucida ◽  
Low Molecular Weight ◽  
Sperm Capacitation ◽  
Vitro Fertilization ◽  
F9 Cells ◽  
Sperm Surface ◽  
Sperm Binding ◽  
Fertilizing Ability

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as "decapacitation factors" when added back to in vitro fertilization assays. These glycoside "decapacitation factors" inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces "decapacitation factio" competition. On the other hand "conventional" low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of "decapacitation factos" is a necessary prerequisite for sperm binding to the zona pellucida.

scholarly journals Streptococcus pneumoniae Choline-Binding Protein E Interaction with Plasminogen/Plasmin Stimulates Migration across the Extracellular Matrix

2007 ◽  
Vol 76 (2) ◽  
pp. 466-476 ◽  
Author(s):  
Cécile Attali ◽  
Cécile Frolet ◽  
Claire Durmort ◽  
Julien Offant ◽  
Thierry Vernet ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Streptococcus Pneumoniae ◽  
Solid Phase ◽  
Binding Protein ◽  
Physiological Role ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Solid Phase Binding ◽  
Vitro Model

ABSTRACT The virulence mechanisms leading Streptococcus pneumoniae to convert from nasopharyngeal colonization to a tissue-invasive phenotype are still largely unknown. Proliferation of infection requires penetration of the extracellular matrix, which occurs by recruitment of host proteases to the bacterial cell surface. We present evidence supporting the role of choline-binding protein E (CBPE) (a member of the surface-exposed choline-binding protein family) as an important receptor for human plasminogen, the precursor of plasmin. The results of ligand overlay blot analyses, solid-phase binding assays, and surface plasmon resonance experiments support the idea of an interaction between CBPE and plasminogen. We have shown that the phosphorylcholine esterase (Pce) domain of CBPE interacts with the plasminogen kringle domains. Analysis of the crystal structure of the Pce domain, followed by site-directed mutagenesis, allowed the identification of the plasminogen-binding region composed in part by lysine residues, some of which map in a linear fashion on the surface of the Pce domain. The biological relevance of the CBPE-plasminogen interaction is supported by the fact that, compared to the wild-type strain, a mutant of pneumococcus with the cbpE gene deleted (i) displays a reduced level of plasminogen binding and plasmin activation and (ii) shows reduced ability to cross the extracellular matrix in an in vitro model. These results support the idea of a physiological role for the CBPE-plasminogen interaction in pneumococcal dissemination into human tissue.

Use of oocytes that failed to be fertilized in vitro to study human sperm-oocyte interactions: comparison of sperm-oolemma and sperm-zona pellucida binding, and relationship with results of IVF

1990 ◽  
Vol 2 (6) ◽  
pp. 641 ◽  
Author(s):  
DY Liu ◽  
A Lopata ◽  
HW Baker
Keyword(s):  
Zona Pellucida ◽  
Logistic Regression Model ◽  
Sperm Concentration ◽  
Human Sperm ◽  
Fertilization In Vitro ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
Binding Ratio ◽  
Sperm Binding

A test for human sperm binding to the oolemma was developed with oocytes that failed to be fertilized in vitro. The zonae pellucidae of the oocytes were removed under a dissecting microscope by brief exposure to dilute HCl (pH 2.5-3.0) in 0.9% NaCl. The zona-free oocytes (ZFOs) were incubated with a mixture of equal numbers of motile sperm from men to be tested and fertile donors. The sperm was differentially labelled with fluorescein or rhodamine and the results expressed as a ratio of the number of test to control sperm bound to several ZFOs in order to control for variability in the ability of the oolemma to bind sperm. The number of sperm bound to the oolemma increased with time and sperm concentration. The sperm-oolemma binding ratio determined for 32 patients undergoing in vitro fertilization (IVF) was significantly correlated with the sperm-zona pellucida (ZP) binding ratio but was not correlated with other sperm tests. The sperm-oolemma binding ratio was also related to the IVF rate, but this was not significant if the sperm-ZP binding ratio was included in the logistic regression model. Only four of the 32 patients had failure of fertilization in vitro. The human sperm-oolemma binding test may be useful for studying the interaction between gametes, but the test is unlikely to be as useful clinically as the sperm-ZP binding test for predicting fertilization in vitro.

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