180. CHARACTERIZATION AND FUNCTION OF THE BULL SPERM PROTEIN Spam1: TWO DISTINCT ISOFORMS WITH DISTINCT ROLES

2009 ◽  
Vol 21 (9) ◽  
pp. 98
Author(s):  
G. Morin ◽  
R. Sullivan ◽  
I. Laflamme ◽  
C. Robert ◽  
P. Leclerc
Keyword(s):  
Transmembrane Domain ◽  
Inhibitory Effect ◽  
Anterior Region ◽  
Protein Orientation ◽  
Sperm Protein ◽  
Sperm Binding ◽  
Glycosylation Sites ◽  
Sperm Membrane ◽  
Bull Sperm ◽  
And Function

We identified an 80 kDa bull sperm protein (p80) that possesses homology with the Sperm adhesion molecule 1 (Spam1), a GPI-anchored glycoprotein conserved amongst mammals that is required for fertilization. Since bovine Spam1 had not been identified, the aim of this project was to determine if p80 is the bovine Spam1 and to test the hypothesis that it plays a role in gamete interaction during bovine fertilization. Amino acid sequence deduced from 3`/5`Race confirmed that homology between p80 and Spam1 in various species ranged from 47 to 61%.It also revealed specific differences including the absence of a GPI-anchor, the presence of a transmembrane domain, and N- and O- glycosylation sites. By generating and characterising antibodies against p80 N- and C-terminal domains, the protein orientation in the sperm membrane was evaluated. We identified two populations of p80 on the sperm head: one internalised in the anterior region and the second localised on the post-acrosomal region with its hyaluronidase domain exposed to the extracellular environment. Proteomic and immunologic analyses revealed that the p80 post-acrosomal population is a shorter isoform originating from the epididymis while the full length p80 located on the anterior region originates from the testis. Finally, the potential function of p80 during the sperm/zp interaction was evaluated by sperm/zona pellucida (zp) binding assay. The C-terminal extremity of p80 was implicated in sperm binding to the zp by antibody and native protein competition. Furthermore, glycosylation was not required during this interaction since deglycosylated p80 in the incubation medium had the same inhibitory effect on zona binding as the native p80. Collectively, the results demonstrated that p80 is the bovine Spam1, and that two isoforms are present on bull sperm. The hyaluronidase activity of the post-acrosomal isoform is required for cumulus penetration, while the other one participates in sperm/zp binding during fertilization.

A Mucin-Specific Protease Enables Molecular and Functional Analysis of Human Cancer-Associated Mucins

2018 ◽  
Author(s):  
Stacy A. Malaker ◽  
Kayvon Pedram ◽  
Michael J. Ferracane ◽  
Elliot C. Woods ◽  
Jessica Kramer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
High Resolution Mass Spectrometry ◽  
Human Cancer ◽  
Glycosylation Sites ◽  
Bacterial Protease ◽  
Specific Protease ◽  
To Come ◽  
And Function

<div> <div> <div> <p>Mucins are a class of highly O-glycosylated proteins that are ubiquitously expressed on cellular surfaces and are important for human health, especially in the context of carcinomas. However, the molecular mechanisms by which aberrant mucin structures lead to tumor progression and immune evasion have been slow to come to light, in part because methods for selective mucin degradation are lacking. Here we employ high resolution mass spectrometry, polymer synthesis, and computational peptide docking to demonstrate that a bacterial protease, called StcE, cleaves mucin domains by recognizing a discrete peptide-, glycan-, and secondary structure- based motif. We exploited StcE’s unique properties to map glycosylation sites and structures of purified and recombinant human mucins by mass spectrometry. As well, we found that StcE will digest cancer-associated mucins from cultured cells and from ovarian cancer patient-derived ascites fluid. Finally, using StcE we discovered that Siglec-7, a glyco-immune checkpoint receptor, specifically binds sialomucins as biological ligands, whereas the related Siglec-9 receptor does not. Mucin-specific proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of glycoprotein structure and function and for deorphanizing mucin-binding receptors. </p> </div> </div> </div>

Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels in the Cardiovascular System

2004 ◽  
Vol 101 (2) ◽  
pp. 390-398 ◽  
Author(s):  
Takashi Kawano ◽  
Shuzo Oshita ◽  
Akira Takahashi ◽  
Yasuo Tsutsumi ◽  
Yoshinobu Tomiyama ◽  
...  
Keyword(s):  
Cardiovascular System ◽  
Adenosine Triphosphate ◽  
Local Anesthetics ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Katp Channels ◽  
Inhibitory Effect ◽  
Amino Acid Residues ◽  
Sulfonylurea Receptor ◽  
Inhibitory Effects

Background Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits. Methods The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs. Results Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit. Conclusions Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.

scholarly journals Acetylcholine Receptor Gating at Extracellular Transmembrane Domain Interface: the “Pre-M1” Linker

2007 ◽  
Vol 130 (6) ◽  
pp. 559-568 ◽  
Author(s):  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
State Energy ◽  
Transmembrane Domain ◽  
Salt Bridge ◽  
Side Chain ◽  
Coupling Energy ◽  
And Function ◽  
Loop 2 ◽  
Α Subunits ◽  
Α1 Subunit

Charged residues in the β10–M1 linker region (“pre-M1”) are important in the expression and function of neuromuscular acetylcholine receptors (AChRs). The perturbation of a salt bridge between pre-M1 residue R209 and loop 2 residue E45 has been proposed as being a principle event in the AChR gating conformational “wave.” We examined the effects of mutations to all five residues in pre-M1 (positions M207–P211) plus E45 in loop 2 in the mouse α1-subunit. M207, Q208, and P211 mutants caused small (approximately threefold) changes in the gating equilibrium constant (Keq), but the changes for R209, L210, and E45 were larger. Of 19 different side chain substitutions at R209 on the wild-type background, only Q, K, and H generated functional channels, with the largest change in Keq (67-fold) from R209Q. Various R209 mutants were functional on different E45 backgrounds: H, Q, and K (E45A), H, A, N, and Q (E45R), and K, A, and N (E45L). Φ values for R209 (on the E45A background), L210, and E45 were 0.74, 0.35, and 0.80, respectively. Φ values for R209 on the wt and three other backgrounds could not be estimated because of scatter. The average coupling energy between 209/45 side chains (six different pairs) was only −0.33 kcal/mol (for both α subunits, combined). Pre-M1 residues are important for expression of functional channels and participate in gating, but the relatively modest changes in closed- vs. open-state energy caused mutations, the weak coupling energy between these residues and the functional activity of several unmatched-charge pairs are not consistent with the perturbation of a salt bridge between R209 and E45 playing the principle role in gating.

scholarly journals Saccharomyces cerevisiae Grx6 and Grx7 Are Monothiol Glutaredoxins Associated with the Early Secretory Pathway

2008 ◽  
Vol 7 (8) ◽  
pp. 1415-1426 ◽  
Author(s):  
Alicia Izquierdo ◽  
Celia Casas ◽  
Ulrich Mühlenhoff ◽  
Christopher Horst Lillig ◽  
Enrique Herrero
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Secretory Pathway ◽  
Transmembrane Domain ◽  
Inhibitory Effect ◽  
Protein Accumulation ◽  
Oxidoreductase Activity ◽  
Early Secretory Pathway ◽  
Glycosylation Inhibitor

ABSTRACT Saccharomyces cerevisiae Grx6 and Grx7 are two monothiol glutaredoxins whose active-site sequences (CSYS and CPYS, respectively) are reminiscent of the CPYC active-site sequence of classical dithiol glutaredoxins. Both proteins contain an N-terminal transmembrane domain which is responsible for their association to membranes of the early secretory pathway vesicles, facing the luminal side. Thus, Grx6 localizes at the endoplasmic reticulum and Golgi compartments, while Grx7 is mostly at the Golgi. Expression of GRX6 is modestly upregulated by several stresses (calcium, sodium, and peroxides) in a manner dependent on the Crz1-calcineurin pathway. Some of these stresses also upregulate GRX7 expression under the control of the Msn2/4 transcription factor. The N glycosylation inhibitor tunicamycin induces the expression of both genes along with protein accumulation. Mutants lacking both glutaredoxins display reduced sensitivity to tunicamycin, although the drug is still able to manifest its inhibitory effect on a reporter glycoprotein. Grx6 and Grx7 have measurable oxidoreductase activity in vivo, which is increased in the presence of tunicamycin. Both glutaredoxins could be responsible for the regulation of the sulfhydryl oxidative state at the oxidant conditions of the early secretory pathway vesicles. However, the differences in location and expression responses against stresses suggest that their functions are not totally overlapping.

scholarly journals Metabolic aspects of the secretion of stored compounds from blood platelets. The effect of NaF at different pH on nucleotide metabolism and function of washed platelets

1981 ◽  
Vol 194 (1) ◽  
pp. 187-192 ◽  
Author(s):  
E H Mürer ◽  
K Davenport ◽  
E Siojo ◽  
H J Day
Keyword(s):  
Time Course ◽  
Adenine Nucleotides ◽  
Blood Platelets ◽  
Fluoride Concentration ◽  
Indirect Evidence ◽  
Inhibitory Effect ◽  
Nucleotide Metabolism ◽  
Wide Range ◽  
And Function ◽  
Special Circumstances

The purpose of this study was to investigate the response of human blood platelets to fluoride at different pH. The results were as follows. (1) Fluoride induced secretion faster and at a lower concentration when pH was lowered. (2) Platelets exposed to 2 mM-fluoride at 0 degrees C at pH 5.3 underwent secretion when first pH and then temperature was raised, although no secretion was seen at 2 mM-fluoride concentration in the absence of the preincubation at low pH. (3) The concentration of [14C]ATP in platelets decreased steeply in response to fluoride before induction of secretion. Addition of antimycin blocked or partly inhibited secretion. Fluoride thus exerts an inhibitory effect on platelet glycolysis before induction of secretion. (4) Fluoride accumulated in the platelet pellet by a time course that preceded secretion. The accumulation was faster and greater at pH 6 than at 7.4. These four points are taken as indirect evidence that fluoride has to penetrate to the interior of the platelet to induce secretion. The activation takes place over a wide range of acid pH in contrast with induction of platelet function via the outside of the plasma membrane. In addition evidence is presented that the salvage pathway may under special circumstances play an important role in the re-synthesis of platelet adenine nucleotides.

scholarly journals The role of prolines and glycine in the transmembrane domain of LAT

2020 ◽  
Author(s):  
Daniela Glatzová ◽  
Harsha Mavila ◽  
Maria Chiara Saija ◽  
Tomáš Chum ◽  
Lukasz Cwiklik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Helical Structure ◽  
Surface Expression ◽  
Transmembrane Segment ◽  
Proline Residue ◽  
And Function ◽  
The Impact

ABSTRACTLAT is a critical regulator of T cell development and function. It organises signalling events at the plasma membrane. However, the mechanism, which controls LAT localisation at the plasma membrane is not fully understood. Here, we studied the impact of helix-breaking amino acids, two prolines and one glycine, in the transmembrane segment on localisation and function of LAT. Using in silico analysis, confocal and superresolution imaging and flow cytometry we demonstrate that central proline residue destabilises transmembrane helix by inducing a kink. The helical structure and dynamics is further regulated by glycine and another proline residue in the luminal part of LAT transmembrane domain. Replacement of these residues with aliphatic amino acids reduces LAT dependence on palmitoylation for sorting to the plasma membrane. However, surface expression of these mutants is not sufficient to recover function of non-palmitoylated LAT in stimulated T cells. These data indicate that geometry and dynamics of LAT transmembrane segment regulate its localisation and function in immune cells.

scholarly journals αIIbβ3 variants in ten families with autosomal dominant macrothrombocytopenia: Expanding the mutational and clinical spectrum

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0235136
Author(s):  
Sara Morais ◽  
Jorge Oliveira ◽  
Catarina Lau ◽  
Mónica Pereira ◽  
Marta Gonçalves ◽  
...  
Keyword(s):  
Binding Sites ◽  
Autosomal Dominant ◽  
Transmembrane Domain ◽  
Laboratory Data ◽  
Atp Release ◽  
Cytoplasmic Domains ◽  
Pathogenic Variants ◽  
Distinct Disease ◽  
And Function ◽  
First Time

Background Rare pathogenic variants in either the ITGA2B or ITGB3 genes have been linked to autosomal dominant macrothrombocytopenia associated with abnormal platelet production and function, deserving the designation of Glanzmann Thrombasthenia-Like Syndrome (GTLS) or ITGA2B/ITGB3-related thrombocytopenia. Objectives To describe a series of patients with familial macrothrombocytopenia and decreased expression of αIIbβ3 integrin due to defects in the ITGA2B or ITGB3 genes. Methods We reviewed the clinical and laboratory records of 10 Portuguese families with GTLS (33 patients and 11 unaffected relatives), including the functional and genetic defects. Results Patients had absent to moderate bleeding, macrothrombocytopenia, low αIIbβ3 expression, impaired platelet aggregation/ATP release to physiological agonists and low expression of activation-induced binding sites on αIIbβ3 (PAC-1) and receptor-induced binding sites on its ligand (bound fibrinogen), upon stimulation with TRAP-6 and ADP. Evidence for constitutive αIIbβ3 activation, occurred in 2 out of 9 patients from 8 families studied, but also in 2 out of 12 healthy controls. We identified 7 missense variants: 3 in ITGA2B (5 families), and 4 in ITGB3 (5 families). Three variants (αIIb: p.Arg1026Trp and p.Arg1026Gln and β3: p.Asp749His) were previously reported. The remaining (αIIb: p.Gly1007Val and β3: p.Thr746Pro, p.His748Pro and p.Arg760Cys) are new, expanding the αIIbβ3 defects associated with GTLS. The integration of the clinical and laboratory data allowed the identification of two GTLS subgroups, with distinct disease severity. Conclusions Previously reported ITGA2B and ITGB3 variants related to thrombocytopenia were clustered in a confined region of the membrane-proximal cytoplasmic domains, the inner membrane clasp. For the first time, variants are reported at the outer membrane clasp, at the transmembrane domain of αIIb, and at the membrane distal cytoplasmic domains of β3. This is the largest single-center series of inherited macrothrombocytopenia associated with αIIbβ3 variants published to date.

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