scholarly journals A common single nucleotide polymorphism A118G of the μ opioid receptor alters its N-glycosylation and protein stability

2011 ◽  
Vol 441 (1) ◽  
pp. 379-386 ◽  
Author(s):  
Peng Huang ◽  
Chongguang Chen ◽  
Stephen D. Mague ◽  
Julie A. Blendy ◽  
Lee-Yuan Liu-Chen
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Amino Acid ◽  
Protein Stability ◽  
Molecular Mass ◽  
Opioid Receptor ◽  
Amino Acid Substitution ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
118G Allele ◽  
Μ Opioid Receptor

The A118G SNP (single nucleotide polymorphism) of the hMOPR [human MOPR (μ opioid receptor)] gene OPRM1 results in an amino acid substitution (N40D). Subjects homozygous for the 118G allele have been reported to require higher morphine doses to achieve adequate analgesia, and the 118G allele is more prevalent among drug abusers. However, changes in the MOPR protein associated with this SNP are unknown. Using a knockin mouse model (G/G mice; mice homozygous for the 112G allele of MOPR) that possesses the equivalent nucleotide/amino acid substitution (A112G; N38D) of the A118G SNP in the hMOPR gene, we investigated the N-linked glycosylation status of thalamic and striatal MOPR in G/G mice compared with A/A mice (wild-type mice homozygous for the 112A allele of MOPR). The molecular mass of MOPR determined by immunoblotting was lower in G/G mice than in A/A mice. Following treatment with peptide N-glycosidase F, which removes all N-linked glycans, both MOPR variants had an identical molecular mass, indicating that this discrepancy was due to a lower level of N-glycosylation of the MOPR in G/G mice. In Chinese-hamster ovary cells stably expressing hMOPRs, 118G/Asp40-hMOPR had a lower molecular mass than 118A/Asn40-hMOPR, which was similarly due to differential N-glycosylation. Pulse–chase studies revealed that the half-life of the mature form of 118G/Asp40-hMOPR (~12 h) was shorter than that of 118A/Asn40-hMOPR (~28 h). Thus the A118G SNP reduces MOPR N-glycosylation and protein stability.

scholarly journals The neutrophil alloantigen HNA-3a (5b) is located on choline transporter-like protein 2 and appears to be encoded by an R>Q154 amino acid substitution

Blood ◽  
2010 ◽  
Vol 115 (10) ◽  
pp. 2073-2076 ◽  
Author(s):  
Brian R. Curtis ◽  
Nancy J. Cox ◽  
Mia J. Sullivan ◽  
Anuar Konkashbaev ◽  
Krista Bowens ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Amino Acid ◽  
Lung Injury ◽  
Amino Acid Substitution ◽  
Large Scale ◽  
Spectrometric Analysis ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Choline Transporter ◽  
Specific Antibodies

Abstract The molecular basis of the HNA-3a/b (5b/a) leukocyte antigen system has not yet been defined despite evidence that HNA-3a–specific antibodies are particularly prone to cause severe, often fatal, transfusion-related lung injury. We used genome-wide single nucleotide polymorphism scanning and sequencing of DNA from persons of different HNA-3a/b phenotypes to identify a single single nucleotide polymorphism in exon 7 of the CLT2 gene (SLC44A2) that predicts an amino acid substitution in the first extracellular loop of choline transporter-like protein 2, a member of the choline transporter-like protein family of membrane glycoproteins, and correlates perfectly with HNA-3a/b phenotypes (R154 encodes HNA-3a; Q154 encodes HNA-3b). Mass spectrometric analysis of proteins immunoprecipitated from leukocytes by anti–HNA-3a provided direct evidence that anti–HNA-3a recognizes choline transporter-like protein 2. These findings will enable large-scale genotyping for HNA-3a/b to identify blood donors at risk to have HNA-3a–specific antibodies and should facilitate development of practical methods to detect such antibodies and prevent transfusion-related lung injury.

Characterization of the Human Neutrophil Alloantigen (HNA) 3a.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 24-24 ◽  
Author(s):  
Juergen Bux ◽  
Jan Wesche ◽  
Elke Hammer ◽  
Uwe Voelker ◽  
Angelika Reil ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Human Neutrophil ◽  
Plasma Samples ◽  
Nucleotide Polymorphism ◽  
Protein Database ◽  
Single Nucleotide ◽  
Choline Transporter

Abstract Abstract 24 Transfusion-related acute lung injury (TRALI) has been recognized as a frequent cause of transfusion-associated major morbidity and mortality in the Western world. About 80% of reported TRALI cases have been associated with the transfusion of blood products containing leukocyte alloantibodies (Middelburg et al., 2008). Up to 28% of severe and fatal TRALI cases have been reported to be associated with antibodies directed against the human neutrophil alloantigen (HNA)-3a, previously known as 5b (Reil et al., 2008). Since the membrane molecule bearing the HNA-3a/b polymorphism is still unknown, we initiated a study with the objective of elucidating the molecular basis of HNA-3a. For characterization of HNA-3a, we first precipitated HNA-3a from biotinylated HNA-3a expressing neutrophils by the use of HNA-3a antibodies, cell solubilisation with Triton-X100 and Protein G beads coupled with goat anti-human IgG. After electrophoretic separation of the immunoprecipitated proteins by SDS-PAGE and transfer onto membrane, we observed in Western blot analysis a broad band of 80-100 kDa which shifted to 64 kDa after deglycosylation with the N-glycosidase PNGase F. Control plasma samples and/or the use of HNA-3a negative granulocytes did not show these bands. Immunoprecipitates of non-biotinylated granulocytes were purified by μC18 tips and concentrated by vacuum drying. Peptide mixtures were separated and analysed using ultra high performance liquid chromatography and tandem mass spectrometry. Proteins were identified by aligning all obtained spectra with a protein database. Ten peptides were identified which were only present in the HNA-3a precipitates but not in the control precipitates. These peptides were found to be parts of the choline transporter-like protein 2 (CTL2 ) with six peptides matching to the first of the ten extracellular domains. We then assessed a panel of 54 individuals serologically typed for HNA-3a for nonsynonymous mutations in the gene SLC44A2 encoding CTL2 by sequence-specific PCR. The single nucleotide polymorphism 461 G>A resulting in an amino acid substitution from arginine to glutamine at position 154 was fully concordant with the HNA-3a/b phenotypes of these 54 individuals. In addition 461G (154Arg) representing the HNA-3a allele was always found in patients who developed TRALI due to HNA-3a alloantibody transfusion, whereas 461A (154Gln) representing the HNA-3b allele was solely present in all blood donors who formed HNA-3a alloantibodies. In a population study of 3700 individuals using DNA microarrays, 176 (4.8%) were homozygous for A461 (HNA-3b), 1188 (32.1%) were heterozygous (AG461), and 2336 (63.1%) were homozygous for G461 (HNA-3a). Finally, we expressed parts of the first extracellular domain of CTL2 in E. coli which included the amino acid position 154. After culturing, fusion proteins were isolated from the bacteria and transferred on membrane. By immunoblotting, specific signals were only obtained with anti-HNA-3a antibodies containing plasma samples but not with controls. We conclude that the HNA-3a/b polymorphism is caused by a single nucleotide polymorphism at position 461G>A in the gene encoding the choline transporter-like protein 2 resulting in an arginine (R) to glutamine (Q) amino acid substitution at position 154. This finding provides the basis for the development of assays allowing large scale screening of blood donor samples for the presence of HNA-3a antibodies. Such screening will contribute to the reduction of severe TRALI. Disclosures: No relevant conflicts of interest to declare.

The single nucleotide polymorphism A118G alters functional properties of the human mu opioid receptor

2007 ◽  
Vol 0 (0) ◽  
pp. 070630082917007-??? ◽  
Author(s):  
Thomas Kroslak ◽  
K. Steven LaForge ◽  
Robert J. Gianotti ◽  
Ann Ho ◽  
David A. Nielsen ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Opioid Receptor ◽  
Functional Properties ◽  
Mu Opioid Receptor ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Mu Opioid

scholarly journals A Single Amino Acid Change in the Response Regulator PhoP, Acquired duringYersinia pestisEvolution, Affects PhoP Target Gene Transcription and Polymyxin B Susceptibility

2018 ◽  
Vol 200 (9) ◽  
pp. e00050-18 ◽  
Author(s):  
Hana S. Fukuto ◽  
Viveka Vadyvaloo ◽  
Joseph B. McPhee ◽  
Hendrik N. Poinar ◽  
Edward C. Holmes ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Target Gene ◽  
Response Regulator ◽  
Polymyxin B ◽  
Single Amino Acid ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Virulent Strains

ABSTRACTYersinia pestis, the causative agent of plague, evolved from the closely related pathogenYersinia pseudotuberculosis. During its emergence,Y. pestisis believed to have acquired its unique pathogenic characteristics through numerous gene gains/losses, genomic rearrangements, and single nucleotide polymorphism (SNP) changes. One such SNP creates a single amino acid variation in the DNA binding domain of PhoP, the response regulator in the PhoP/PhoQ two-component system.Y. pseudotuberculosisand the basal human-avirulent strains ofY. pestisharbor glycines at position 215 of PhoP, whereas the modern human-virulent strains (e.g., KIM and CO92) harbor serines at this residue. Since PhoP plays multiple roles in the adaptation ofY. pestisto stressful host conditions, we tested whether this amino acid substitution affects PhoP activity or the ability ofY. pestisto survive in host environments. Compared to the parental KIM6+ strain carrying the modern allele ofphoP(phoP-S215), a derivative carrying the basal allele (phoP-G215) exhibited slightly defective growth under a low-Mg2+condition and decreased transcription of a PhoP target gene,ugd, as well as an ∼8-fold increase in the susceptibility to the antimicrobial peptide polymyxin B. ThephoP-G215strain showed no apparent defect in flea colonization, although aphoP-null mutant showed decreased flea infectivity in competition experiments. Our results suggest that the amino acid variation at position 215 of PhoP causes subtle changes in the PhoP activity and raise the possibility that the change in this residue have contributed to the evolution of increased virulence inY. pestis.IMPORTANCEY. pestisacquired a single nucleotide polymorphism (SNP) inphoPwhen the highly human-virulent strains diverged from less virulent basal strains, resulting in an amino acid substitution in the DNA binding domain of the PhoP response regulator. We show thatY. pestiscarrying the modernphoPallele has an increased ability to induce the PhoP-regulatedugdgene and resist antimicrobial peptides compared to an isogenic strain carrying the basal allele. Given the important roles PhoP plays in host adaptation, the results raise an intriguing possibility that this amino acid substitution contributed to the evolution of increased virulence inY. pestis. Additionally, we present the first evidence thatphoPconfers a survival fitness advantage toY. pestisinside the flea midgut.

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