Residues SFQ (173-175) in the large extracellular loop of CD9 are required for gamete fusion

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1995-2002 ◽  
Author(s):  
Guo-Zhang Zhu ◽  
Brent J. Miller ◽  
Claude Boucheix ◽  
Eric Rubinstein ◽  
Christopher C. Liu ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Point Mutation ◽  
Active Site ◽  
Gene Knockout ◽  
Extracellular Loop ◽  
Gamete Fusion ◽  
Extracellular Loops ◽  
Egg Membrane ◽  
Large Extracellular Loop ◽  
Fusion Ability

Gamete fusion is the fundamental first step initiating development of a new organism. Female mice with a gene knockout for the tetraspanin CD9 (CD9 KO mice) produce mature eggs that cannot fuse with sperm. However, nothing is known about how egg surface CD9 functions in the membrane fusion process. We found that constructs including CD9’s large extracellular loop significantly inhibited gamete fusion when incubated with eggs but not when incubated with sperm, suggesting that CD9 acts by interaction with other proteins in the egg membrane. We also found that injecting developing CD9 KO oocytes with CD9 mRNA restored fusion competence to the resulting CD9 KO eggs. Injecting mRNA for either mouse CD9 or human CD9, whose large extracellular loops differ in 18 residues, rescued fusion ability of the injected CD9 KO eggs. However, when the injected mouse CD9 mRNA contained a point mutation (F174 to A) the gamete fusion level was reduced fourfold, and a change of three residues (173-175, SFQ to AAA) abolished CD9’s activity in gamete fusion. These results suggest that SFQ in the CD9 large extracellular loop may be an active site which associates with and regulates the egg fusion machinery.

Cleavage of SPACA1 regulates assembly of sperm–egg membrane fusion machinery in mature spermatozoa†

2019 ◽  
Vol 102 (3) ◽  
pp. 750-757
Author(s):  
Kenji Yamatoya ◽  
Marika Kousaka ◽  
Chizuru Ito ◽  
Kazuya Nakata ◽  
Masahiko Hatano ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Acrosome Reaction ◽  
Molecular Mechanisms ◽  
Morphological Changes ◽  
Reaction Process ◽  
Related Factor ◽  
Gamete Fusion ◽  
C Terminus ◽  
Egg Membrane ◽  
Mature Spermatozoa

Abstract The acrosome reaction is a multi-step event essential for physiological fertilization. During the acrosome reaction, gamete fusion-related factor IZUMO1 translocates from the anterior acrosome to the equatorial segment and assembles the gamete fusion machinery. The morphological changes in the acrosome reaction process have been well studied, but little is known about the molecular mechanisms of acrosome reorganization essential for physiological gamete membrane fusion. To elucidate the molecular mechanisms of IZUMO1 translocation, the steps of the acrosome reaction during that process must be clarified. In this study, we established a method to detect the early steps of the acrosome reaction and subdivided the process into seven populations through the use of two epitope-defined antibodies, anti-IZUMO1 and anti-SPACA1, a fertilization-inhibiting antibody. We found that part of the SPACA1 C-terminus in the periacrosomal space was cleaved and had begun to disappear when the vesiculation of the anterior acrosome occurred. The IZUMO1 epitope externalized from the acrosomal lumen before acrosomal vesiculation and phosphorylation of IZUMO1 occurred during the translocation to the equatorial segment. IZUMO1 circumvented the area of the equatorial segment where the SPACA1C-terminus was still localized. We therefore propose an IZUMO1 translocation model and involvement of SPACA1.

scholarly journals Large Extracellular Loop of Tetraspanin as a Potential Vaccine Candidate for Filariasis

PLoS ONE ◽  
2013 ◽  
Vol 8 (10) ◽  
pp. e77394 ◽  
Author(s):  
Gajalakshmi Dakshinamoorthy ◽  
Gnanasekar Munirathinam ◽  
Kristen Stoicescu ◽  
Maryada Venkatarami Reddy ◽  
Ramaswamy Kalyanasundaram
Keyword(s):  
Vaccine Candidate ◽  
Extracellular Loop ◽  
Potential Vaccine Candidate ◽  
Large Extracellular Loop ◽  
Potential Vaccine

Sperm-induced local [Ca2+]i rise separated from the Ca2+ wave in sea urchin eggs in the presence of a gamete fusion inhibitor, jaspisin

Development ◽  
1998 ◽  
Vol 125 (2) ◽  
pp. 293-300 ◽  
Author(s):  
T. Mohri ◽  
S. Miyazaki ◽  
H. Shirakawa ◽  
S. Ikegami
Keyword(s):  
Membrane Fusion ◽  
Sea Urchin ◽  
Focal Plane ◽  
Attachment Site ◽  
Fusion Inhibitor ◽  
Low Doses ◽  
Inward Current ◽  
Egg Membrane ◽  
Hemicentrotus Pulcherrimus ◽  
Stimulation Of

An increase in intracellular Ca2+ concentration ([Ca2+]i) at a focal plane was recorded simultaneously with sperm-egg binding and membrane current upon insemination of sea urchin Hemicentrotus pulcherrimus eggs. No change in current and [Ca2+]i occurred in the presence of jaspisin, a novel substance that inhibits metallo-endoproteinase and sperm-egg membrane fusion (S. Ikegami, H. Kobayashi, Y. Myotoishi, S. Ohta and K. H. Kato (1994) J. Biol. Chem. 269, 23262–23267). With low doses of jaspisin, a spermatozoon first produced a step inward current (I(on)) as an indication of gamete membrane fusion and then induced a local [Ca2+]i rise at the site of sperm attachment 6–10 seconds after I(on). The sperm, however, soon detached from the egg. Increasing inward current was abruptly cut off (I(off)) within 9–15 seconds and the local [Ca2+]i rise began to decline 1–3 seconds after I(off). In most cases, no further responses or an elevation of fertilization envelope (FE) occurred. In some cases, [Ca2+]i at the sperm attachment site increased again even after the sperm detached and triggered a Ca2+ wave which caused an activation current and FE formation. This recording of a gamete membrane-fusion-induced local [Ca2+]i rise, separated from the Ca2+ wave, is a key phenomenon for elucidating the initial sperm stimulation of the egg at fertilization.

Kinetics of HCV envelope proteins’ interaction with CD81 large extracellular loop

2005 ◽  
Vol 328 (4) ◽  
pp. 1091-1100 ◽  
Author(s):  
Hideki Nakajima ◽  
Laurence Cocquerel ◽  
Nobutaka Kiyokawa ◽  
Junichiro Fujimoto ◽  
Shoshana Levy
Keyword(s):  
Envelope Proteins ◽  
Extracellular Loop ◽  
Large Extracellular Loop ◽  
Kinetics Of

scholarly journals A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A

2006 ◽  
Vol 87 (1) ◽  
pp. 93-102 ◽  
Author(s):  
Christopher J. McCormick ◽  
David Brown ◽  
Stephen Griffin ◽  
Lisa Challinor ◽  
David J. Rowlands ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Point Mutation ◽  
Active Site ◽  
Natural Infection ◽  
Polymerase Activity ◽  
Encephalomyocarditis Virus ◽  
Structural Proteins ◽  
Hcv Rna ◽  
Viral Transcript

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

scholarly journals Changes to Gonadotropin-Releasing Hormone (GnRH) Receptor Extracellular Loops Differentially Affect GnRH Analog Binding and Activation: Evidence for Distinct Ligand-Stabilized Receptor Conformations

Endocrinology ◽  
2008 ◽  
Vol 149 (6) ◽  
pp. 3118-3129 ◽  
Author(s):  
Kevin D. G. Pfleger ◽  
Adam J. Pawson ◽  
Robert P. Millar
Keyword(s):  
Ligand Binding ◽  
Receptor Activation ◽  
Gnrh Receptor ◽  
Binding Modes ◽  
Extracellular Loop ◽  
Gnrh Analog ◽  
Chimeric Receptors ◽  
Gnrh Receptors ◽  
Extracellular Loops ◽  
Loop Conformation

GnRH and its structural variants bind to GnRH receptors from different species with different affinities and specificities. By investigating chimeric receptors that combine regions of mammalian and nonmammalian GnRH receptors, a greater understanding of how different domains influence ligand binding and receptor activation can be achieved. Using human-catfish and human-chicken chimeric receptors, we demonstrate the importance of extracellular loop conformation for ligand binding and agonist potency, providing further evidence for GnRH and GnRH II stabilization of distinct active receptor conformations. We demonstrate examples of GnRH receptor gain-of-function mutations that apparently improve agonist potency independently of affinity, implicating a role for extracellular loops in stabilizing the inactive receptor conformation. We also show that entire extracellular loop substitution can overcome the detrimental effects of localized mutations, thereby demonstrating the importance of considering the conformation of entire domains when drawing conclusions from point-mutation studies. Finally, we present evidence implicating the configuration of extracellular loops 2 and 3 in combination differentiating GnRH analog binding modes. Because there are two endogenous forms of GnRH ligand but only one functional form of full-length GnRH receptor in humans, understanding how GnRH and GnRH II can elicit distinct functional effects through the same receptor is likely to provide important insights into how these ligands can have differential effects in both physiological and pathological situations.

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