scholarly journals Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins

1991 ◽  
Vol 273 (2) ◽  
pp. 301-301 ◽  
Author(s):  
N M Hooper ◽  
S J Broomfield ◽  
A J Turner
Keyword(s):  
Phospholipase C ◽  
Cross Reactivity ◽  
Side Chain ◽  
Glycosyl Phosphatidylinositol ◽  
Anchor Structure ◽  
Polyclonal Antisera ◽  
Aminopeptidase P ◽  
Species Specific ◽  
Mild Acid

Two polyclonal antisera were raised in rabbits to the phospholipase C-solubilized forms of pig renal dipeptidase (EC 3.4.13.11) and pig aminopeptidase P (EC 3.4.11.9). These antisera were purified and shown to cross-react with other glycosyl-phosphatidylinositol (G-PI)-anchored proteins isolated from pig, human and trypanosomes. The epitopes involved in this cross-reactivity were characterized by Western-blot analysis after mild acid or nitrous acid treatment of the G-PI-anchored proteins and by a competitive e.l.i.s.a. with other G-PI-anchored proteins and individual components of the anchor structure. These studies revealed that the primary epitope for both antisera is the inositol 1.2-(cyclic)monophosphate that is formed on phospholipase C cleavage of the intact G-PI anchor. Other minor epitopes, such as phosphoethanolamine, probably involve side-chain modifications to the core anchor structure that may be species-specific.

scholarly journals Purification and characterization of pig kidney aminopeptidase P. A glycosyl-phosphatidylinositol-anchored ectoenzyme

1990 ◽  
Vol 267 (2) ◽  
pp. 509-515 ◽  
Author(s):  
N M Hooper ◽  
J Hryszko ◽  
A J Turner
Keyword(s):  
Chelating Agents ◽  
Inhibitory Effect ◽  
Purification And Characterization ◽  
Glycosyl Phosphatidylinositol ◽  
Pig Kidney ◽  
Sds Page ◽  
Cyclic Phosphate ◽  
Aminopeptidase P ◽  
Mild Acid

Aminopeptidase P (EC 3.4.11.9) was solubilized from pig kidney membranes with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) and then purified by a combination of anion-exchange and hydrophobic-interaction chromatographies. Contaminating peptidase activities were removed by selective affinity chromatography. The purified enzyme was apparently homogeneous on SDS/PAGE with an Mr of 91,000. Enzymic deglycosylation revealed that aminopeptidase P is a glycoprotein, with up to 25% by weight of the protein being due to the presence of N-linked sugars. The phospholipase-solubilized aminopeptidase P was recognized by an antiserum to the cross-reacting determinant (CRD) characteristic of the glycosyl-phosphatidylinositol anchor. This recognition was abolished by mild acid treatment or deamination with HNO2, indicating that the CRD was due exclusively to the inositol 1,2-cyclic phosphate ring epitope generated by the action of PI-PLC. The activity of aminopeptidase P was inhibited by chelating agents and was stimulated by Mn2+ or Co2+ ions, confirming the metallo-enzyme nature of this peptidase. Selective inhibitors of other aminopeptidases (actinonin, amastatin, bestatin and puromycin) had little or no inhibitory effect.

scholarly journals Structural characterization of a novel glycosyl-phosphatidylinositol from the protozoan Tetrahymena mimbres

1991 ◽  
Vol 279 (2) ◽  
pp. 605-608 ◽  
Author(s):  
U Weinhart ◽  
J R Thomas ◽  
Y Pak ◽  
G A Thompson ◽  
M A J Ferguson
Keyword(s):  
Phospholipase C ◽  
Structural Characterization ◽  
Protozoan Parasites ◽  
Free Living ◽  
Glycosyl Phosphatidylinositol

A glycolipid metabolically labelled with [14C]GlcN was isolated from the free-living protozoan Tetrahymena mimbres. The glycolipid was sensitive to a bacterial phosphatidylinositol-specific phospholipase C, and the headgroup was shown to contain a phosphorylated Man alpha 1-2Man alpha 1-4Man alpha 1-4GlcN glycan. The Tetrahymena glycolipid is structurally unique among the glycosylphosphatidylinositols that have so far been characterized, including those from several protozoan parasites of humans.

scholarly journals Potent human neutralizing antibodies elicited by SARS-CoV-2 infection

2020 ◽  
Author(s):  
Bin Ju ◽  
Qi Zhang ◽  
Xiangyang Ge ◽  
Ruoke Wang ◽  
Jiazhen Yu ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Cell Receptor ◽  
Cross Reactivity ◽  
Therapeutic Interventions ◽  
Global Public Health ◽  
Angiotensin Converting Enzyme 2 ◽  
Isolation And Characterization ◽  
Protein Receptor ◽  
Species Specific

AbstractThe pandemic caused by emerging coronavirus SARS-CoV-2 presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. SARS-CoV-2 cellular entry depends on binding between the viral Spike protein receptor-binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) target cell receptor. Here, we report on the isolation and characterization of 206 RBD-specific monoclonal antibodies (mAbs) derived from single B cells of eight SARS-CoV-2 infected individuals. These mAbs come from diverse families of antibody heavy and light chains without apparent enrichment for particular families in the repertoire. In samples from one patient selected for further analyses, we found coexistence of germline and germline divergent clones. Both clone types demonstrated impressive binding and neutralizing activity against pseudovirus and live SARS-CoV-2. However, the antibody neutralizing potency is determined by competition with ACE2 receptor for RBD binding. Surprisingly, none of the SARS-CoV-2 antibodies nor the infected plasma cross-reacted with RBDs from either SARS-CoV or MERS-CoV although substantial plasma cross-reactivity to the trimeric Spike proteins from SARS-CoV and MERS-CoV was found. These results suggest that antibody response to RBDs is viral species-specific while that cross-recognition target regions outside the RBD. The specificity and neutralizing characteristics of this plasma cross-reactivity requires further investigation. Nevertheless, the diverse and potent neutralizing antibodies identified here are promising candidates for prophylactic and therapeutic SARS-CoV-2 interventions.

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