ABSTRACT
We have induced a polyclonal IgG that degrades the HIV-1 surface antigen, glycoprotein gp120, by taking advantage of the susceptibility of SJL mice to a peptide-induced autoimmune disorder, experimental autoimmune encephalomyelitis (EAE). Specific pathogen-free SJL mice were immunized with structural fragments of gp120, fused in-frame with encephalitogenic peptide MBP(85-101). It has resulted in a pronounced disease-associated immune response against antigens. A dramatic increase of gp120 degradation level by purified polyclonal IgG from immunized versus nonimmunized mice has been demonstrated by a newly developed fluorescence-based assay. This activity was inhibited by anti-mouse immunoglobulin antibodies as well as by Ser- and His-reactive covalent inhibitors. A dominant proteolysis site in recombinant gp120 incubated with purified polyclonal IgG from immunized mice was shown by SDS-PAGE. The SELDI-based mass spectrometry revealed that these antibodies exhibited significant specificity toward the Pro484-Leu485 peptide bond. The sequence surrounding this site is present in nearly half of the HIV-I variants. This novel strategy can be generalized for creating a catalytic vaccine against viral pathogens.
Subject(s)
Antibodies, Catalytic/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , HIV Envelope Protein gp120/metabolism , HIV-1/chemistry , Animals , Antibody Affinity , Autoimmune Diseases/chemically induced , Autoimmune Diseases/immunology , Catalysis , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes/chemistry , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Leucine/chemistry , Mice , Peptide Fragments/chemistry , Peptide Fragments/immunology , Proline/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Viral Vaccines/immunologyABSTRACT
Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.
Subject(s)
Antigens/immunology , Antigens/metabolism , Autoantibodies/immunology , Myelin Basic Protein/immunology , Myelin Basic Protein/metabolism , Amino Acid Sequence , Animals , Antigens/chemistry , Autoantibodies/blood , Catalytic Domain , Encephalomyelitis, Autoimmune, Experimental/immunology , Humans , Mass Spectrometry , Mice , Mice, Inbred C57BL , Multiple Sclerosis/immunology , Multiple Sclerosis/pathology , Myelin Basic Protein/chemistry , Substrate SpecificityABSTRACT
The 70-kDa heat shock protein (Hsp) family in all Drosophila species includes 2 environmentally inducible family members, Hsp70 and Hsp68. Two-dimensional gel electrophoresis revealed an unusual pattern of heat shock-inducible proteins in the species of the virilis group. Trypsin fingerprinting and microsequencing of tryptic peptides using ProteinChip Array technology identified the major isoelectric variants of Hsp70 family, including Hsp68 isoforms that differ in both molecular mass and isoelectric point from those in Drosophila melanogaster. The peculiar electrophoretic mobility is consistent with the deduced amino acid sequence of corresponding hsp genes from the species of the virilis group.
Subject(s)
Drosophila/classification , Drosophila/genetics , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Animals , Antibodies, Monoclonal/metabolism , Electrophoresis, Gel, Two-Dimensional , Electrophoresis, Polyacrylamide Gel , Genetic Variation , Heat-Shock Response/genetics , Hot Temperature , Immunoblotting , Molecular Weight , Peptide Mapping , Protein Isoforms/genetics , Protein Isoforms/metabolism , Sequence Analysis, DNA , Species Specificity , Trypsin/pharmacologyABSTRACT
The composition of RNase H2 has been a long-standing problem. Whereas bacterial and archaeal RNases H2 are active as single polypeptides, the Saccharomyces cerevisiae homolog, Rnh2Ap, when expressed in Escherichia coli, fails to produce an active RNase H2. By affinity chromatography purification and identification of polypeptides associated with a tagged S.cerevisiae Rnh2Ap, we obtained a complex of three proteins [Rnh2Ap (Rnh201p), Ydr279p (Rnh202p) and Ylr154p (Rnh203p)] that together are necessary and sufficient for RNase H2 activity [correction]. Deletion of the gene encoding any one of the proteins or mutations in the catalytic site in Rnh2A led to loss of RNase H2 activity. Even when S.cerevisiae RNase H2 is catalytically compromised, it still exhibits a preference for cleavage of the phosphodiester bond on the 5' side of a ribonucleotide-deoxyribonucleotide sequence in substrates mimicking RNA-primed Okazaki fragments or a single ribonucleotide embedded in a duplex DNA. Interestingly, Ydr279p and Ylr154p have homologous proteins only in closely related species. The multisubunit nature of S.cerevisiae RNase H2 may be important both for structural purposes and to provide a means of interacting with other proteins involved in DNA replication/repair and transcription.
