Mouse sperm-egg plasma membrane interactions: analysis of roles of egg integrins and the mouse sperm homologue of PH-30 (fertilin) beta

1995 ◽  
Vol 108 (10) ◽  
pp. 3267-3278 ◽  
Author(s):  
J.P. Evans ◽  
R.M. Schultz ◽  
G.S. Kopf
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Guinea Pig ◽  
Ligand Binding ◽  
Zona Pellucida ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Rgd Peptides ◽  
Mouse Sperm ◽  
Disintegrin Domain

The guinea pig sperm protein, PH-30 (also known as fertilin), is postulated to participate in the interaction between the sperm and egg plasma membranes. The beta subunit of guinea pig PH-30 (gpPH-30 beta) contains a domain with homology to disintegrins, snake venom proteins that bind to integrins via an integrin-binding domain containing the tripeptide RGD. This raises the question of whether an egg integrin serves as a receptor for PH-30. Although mouse eggs express integrin subunits, their role in mouse fertilization is unresolved. Therefore, we examined fertilization for two different hallmarks of integrin function, namely, dependence of ligand binding on divalent cations and the ability to inhibit ligand binding with RGD peptides. We demonstrate that sperm binding to zona pellucida-free eggs is supported by Ca2+, Mg2+, or Mn2+. Ca2+ was necessary and sufficient for sperm-egg fusion, with 2.5 mM Ca2+ being the most effective concentration. In addition, fertilization could be partially inhibited with various RGD peptides, which caused a decrease in sperm-egg fusion by 30–58%. This partial inhibition of fusion with RGD peptides prompted the cloning of the mouse homologue of gpPH-30 beta (hereafter referred to as mPH-30 beta) to determine if it possessed the tripeptide RGD or a different amino acid sequence in its disintegrin domain. mPH-30 beta, which is expressed during meiotic and post-meiotic phases of spermatogenesis, shares significant similarities to gpPH-30 beta throughout the length of the molecule, from the signal sequence to the cytoplasmic tail. The full-length deduced amino acid sequence of mPH-30 beta. The disintegrin domain of mPH-30 beta has the tripeptide QDE (instead of RGD) in its cell recognition region. Peptides containing this QDE sequence decrease the binding and fusion of sperm with zona pellucida-free eggs by approximately 70%, suggesting that the disintegrin domain of mPH-30 beta participates in the interaction between sperm and egg membranes.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

Amino acid sequence of the AhR1 ligand-binding domain predicts avian sensitivity to dioxin like compounds: In vivo verification in European starlings

2014 ◽  
Vol 33 (12) ◽  
pp. 2753-2758 ◽  
Author(s):  
Margaret L. Eng ◽  
John E. Elliott ◽  
Stephanie P. Jones ◽  
Tony D. Williams ◽  
Ken G. Drouillard ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ligand Binding ◽  
Binding Domain ◽  
Ligand Binding Domain ◽  
European Starlings

Amino Acid Sequence of Glutathione S-Transferase a from Guinea Pig Liver

1993 ◽  
Vol 114 (6) ◽  
pp. 835-841
Author(s):  
Raeko Kamei-Hayashi ◽  
Reiko Oshino ◽  
Saburo Hara
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Guinea Pig ◽  
Glutathione S Transferase ◽  
Pig Liver ◽  
Guinea Pig Liver

scholarly journals Molecular Cloning of the Gene for a Conserved Major Immunoreactive 28-Kilodalton Protein of Ehrlichia canis: a Potential Serodiagnostic Antigen

1999 ◽  
Vol 6 (3) ◽  
pp. 392-399 ◽  
Author(s):  
Jere W. McBride ◽  
Xue-jie Yu ◽  
David H. Walker
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Family ◽  
Signal Sequence ◽  
Ehrlichia Canis ◽  
Variable Regions ◽  
Reading Frame ◽  
Cowdria Ruminantium ◽  
Canine Ehrlichiosis ◽  
Gene Copies

ABSTRACT A gene encoding a 28-kDa protein of Ehrlichia canis was cloned, sequenced, and expressed, and a comparative molecular analysis with homologous genes of E. canis, Cowdria ruminantium, and Ehrlichia chaffeensis was performed. The complete gene has an 834-bp open reading frame encoding a protein of 278 amino acids with a predicted molecular mass of 30.5 kDa. An N-terminal signal sequence was identified, suggesting that the protein undergoes posttranslational modification to a mature 27.7-kDa protein (P28). The E. canis p28 gene has significant nucleic acid and amino acid sequence homologies with the E. chaffeensisouter membrane protein-1 (omp-1) gene family, with theCowdria ruminantium map-1 gene, and with other E. canis 28-kDa-protein genes. Southern blotting revealed the presence of at least two additional homologous p28 gene copies in the E. canis genome, confirming thatp28 is a member of a polymorphic multiple-gene family. Amino acid sequence analysis revealed that E. canis P28 has four variable regions, and it shares similar surface-exposed regions, antigenicity, and T-cell motifs with E. chaffeensis P28. The p28 genes from seven different E. canisisolates were identical, indicating that the gene for this major immunoreactive protein is highly conserved. In addition, reactivity of sera from clinical cases of canine ehrlichiosis with the recombinant P28 demonstrated that the recombinant protein may be a reliable serodiagnostic antigen.

scholarly journals Ponticulin is an atypical membrane protein.

1994 ◽  
Vol 126 (6) ◽  
pp. 1421-1431 ◽  
Author(s):  
A L Hitt ◽  
T H Lu ◽  
E J Luna
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Signal Sequence ◽  
Metabolic Labeling ◽  
Cortical Actin ◽  
Intact Cells ◽  
Membrane Spanning

We have cloned and sequenced ponticulin, a 17,000-dalton integral membrane glycoprotein that binds F-actin and nucleates actin assembly. A single copy gene encodes a developmentally regulated message that is high during growth and early development, but drops precipitously during cell streaming at approximately 8 h of development. The deduced amino acid sequence predicts a protein with a cleaved NH2-terminal signal sequence and a COOH-terminal glycosyl anchor. These predictions are supported by amino acid sequencing of mature ponticulin and metabolic labeling with glycosyl anchor components. Although no alpha-helical membrane-spanning domains are apparent, several hydrophobic and/or sided beta-strands, each long enough to traverse the membrane, are predicted. Although its location on the primary sequence is unclear, an intracellular domain is indicated by the existence of a discontinuous epitope that is accessible to antibody in plasma membranes and permeabilized cells, but not in intact cells. Such a cytoplasmically oriented domain also is required for the demonstrated role of ponticulin in binding actin to the plasma membrane in vivo and in vitro (Hitt, A. L., J. H. Hartwig, and E. J. Luna. 1994. Ponticulin is the major high affinity link between the plasma membrane and the cortical actin network in Dictyostelium. J. Cell Biol. 126:1433-1444). Thus, ponticulin apparently represents a new category of integral membrane proteins that consists of proteins with both a glycosyl anchor and membrane-spanning peptide domain(s).

scholarly journals Structure of a group C streptococcal protein that binds to fibrinogen, albumin and immunoglobulin G via overlapping modules

1996 ◽  
Vol 315 (2) ◽  
pp. 577-582 ◽  
Author(s):  
Susanne R. TALAY ◽  
Melanie P. GRAMMEL ◽  
Gursharan S. CHHATWAL
Keyword(s):  
Amino Acid ◽  
Ligand Binding ◽  
Serum Proteins ◽  
Signal Sequence ◽  
Binding Activity ◽  
Binding Domain ◽  
Surface Proteins ◽  
Albumin Binding ◽  
Igg Binding ◽  
Multiple Ligand

Pathogenic streptococci express surface proteins that bind to host serum proteins. A novel multiple-ligand-binding protein has now been identified in a species belonging to serotype C streptococci. This protein binds to fibrinogen, albumin and IgG and was therefore designated FAI protein. The structure of the fai gene has been determined, and deletion analysis and expression of FAI fusion polypeptides revealed that the binding domain for fibrinogen and IgG is located within the non-repetitive N-terminal half of the protein. A 93-amino acid peptide retained the ability to bind both proteins, whereas a 56-amino acid subpeptide only bound fibrinogen. IgG-binding activity required the complete fibrinogen-binding domain and an additional 37 amino acids C-terminal to it, and albumin-binding activity was only obtained with a polypeptide reflecting the complete surface-exposed region of FAI protein indicating that the binding sites for each ligand were located on overlapping modules. Signal sequence, C repeat region and C-terminus revealed high homology to group A streptococcal M proteins whereas the N-terminal region containing the fibrinogen/IgG-binding domains is completely different and exhibits no similarity to any other previously characterized protein. Thus FAI protein exhibits a framework structure that might have evolved in group C streptococci via fusion of unrelated sequences, thereby generating an albumin-binding domain in the functional context of multiple-ligand-binding activity.

Isolation and in vitro translation of mRNA from the calf abomasal mucosa and identification of an mRNA coding for a precursor of prochymosin

1984 ◽  
Vol 51 (1) ◽  
pp. 79-89
Author(s):  
Bruce H. Nicholson ◽  
Peter Jones
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gel Electrophoresis ◽  
Signal Sequence ◽  
In Vitro Translation ◽  
Major Band ◽  
Polyadenylated Rna ◽  
Age Related

SummaryThe mRNA coding for prochymosin (prorennin) has been partly purified from calf abomasum. The in vitro translation products of the total polyadenylated RNA show a major band on gel electrophoresis which reacts with antibody raised against purified chymosin. The mol. wt of 43000 is higher than expected from the reported amino acid sequence and would correspond to prochymosin with an unprocessed signal sequence of ∼ 17 amino acids. The synthesis of chymosin mRNA is age-related, and ceases by 3 months even in milk-fed calves.

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