ABSTRACT
The V3 loop of the HIV-1 envelope (Env) protein elicits a vigorous, but largely non-neutralizing antibody response directed to the V3-crown, whereas rare broadly neutralizing antibodies (bnAbs) target the V3-base. Challenging this view, we present V3-crown directed broadly neutralizing Designed Ankyrin Repeat Proteins (bnDs) matching the breadth of V3-base bnAbs. While most bnAbs target prefusion Env, V3-crown bnDs bind open Env conformations triggered by CD4 engagement. BnDs achieve breadth by focusing on highly conserved residues that are accessible in two distinct V3 conformations, one of which resembles CCR5-bound V3. We further show that these V3-crown conformations can, in principle, be attacked by antibodies. Supporting this conclusion, analysis of antibody binding activity in the Swiss 4.5 K HIV-1 cohort (n = 4,281) revealed a co-evolution of V3-crown reactivities and neutralization breadth. Our results indicate a role of V3-crown responses and its conformational preferences in bnAb development to be considered in preventive and therapeutic approaches.
Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV-1/immunology , Protein Conformation , env Gene Products, Human Immunodeficiency Virus/chemistry , Antibodies, Neutralizing/metabolism , Cell Line, Tumor , Epitopes/genetics , Epitopes/immunology , Epitopes/metabolism , HEK293 Cells , HIV Antibodies/metabolism , HIV-1/genetics , HIV-1/metabolism , Humans , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Binding , env Gene Products, Human Immunodeficiency Virus/genetics , env Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
Gamma-glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathione homeostasis control. It has also been implicated in many physiological disorders, including Parkinson's disease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione, its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. This intermediate can then react with water to release glutamate as a hydrolysis product or with an amino acid or dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying these reactions is hindered at least for some GGTs by the low quantities of protein available after a multistep purification from tissue. In the present work the gene for human GGT was cloned into the pPICZalphaA vector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimized expression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilized metal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg. Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determined by mass spectrometry, giving a kcat of 13.4 x 10(3) min-1 and apparent KM values of 1.11 mM for glutathione and 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providing a kcat of 53 min-1 and a KM value of 7.3 microM for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguous identification of Thr381 as the active site nucleophile of human gamma-glutamyltranspeptidase, and confirmed four of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respect to a homology model generated to facilitate visualization.
Subject(s)
gamma-Glutamyltransferase/metabolism , Acylation , Amino Acid Sequence , Base Sequence , Chromatography, Liquid , DNA Primers , Glycosylation , Humans , Kinetics , Molecular Sequence Data , Pichia/genetics , Polymerase Chain Reaction , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Tandem Mass Spectrometry , gamma-Glutamyltransferase/chemistry , gamma-Glutamyltransferase/isolation & purificationABSTRACT
gamma-Glutamyl transpeptidase (GGT, EC 2.3.2.2) is a highly glycosylated heterodimeric enzyme linked to the external cellular membrane that catalyzes the hydrolysis of glutathione as well as the transfer of its gamma-glutamyl group to amino acids and dipeptides in a transpeptidation reaction. The measurement of both the hydrolytic and transpeptidation activity of this important enzyme is a challenge, since its native substrates are not highly chromogenic. We have developed an HPLC-based method for the quantitative photometric detection of numerous enzyme substrates and products, after their pre-column derivation with dabsyl chloride. The broad applicability of this method was demonstrated in the kinetic investigation of transpeptidation reactions of rat kidney GGT with glutathione, its native substrate, as well as a series of pertinent glutathione analogues. The pH-rate profile constructed for glutathione confirmed the dependence on the ionisation state of at least two residues. Analysis of the free-energy relationships in the series of synthetic peptidic substrate analogues revealed the importance of enzyme-substrate interactions unrelated to amine leaving group basicity during the acylation step. These results are further interpreted in the context of the recently published structure for a similar GGT.
Subject(s)
Peptides/metabolism , gamma-Glutamyltransferase/metabolism , Acylation , Amides/chemical synthesis , Animals , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Glutathione/metabolism , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Peptides/chemical synthesis , Peptides/chemistry , Rats , Substrate Specificity , ThermodynamicsABSTRACT
A new series of L-glutamic acid p-nitroanilide analogues has been synthesized and tested as substrates and inhibitors of rat kidney gamma-glutamyltranspeptidase (GGT). Kinetic parameters (K(m) and k(cat)) were determined for each analogue and provide insight into the scope and limits of GGT catalytic efficiency.
