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Biomedical subjects

A G Plaut

Publications and source records attributed to A G Plaut.

18 recordsLinked to original sources
PubMed1992

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae.

The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.

Amino Acid Sequence↗
PubMed1992

Growth of Haemophilus influenzae in human milk: synthesis, distribution, and activity of IgA protease as determined by study of iga+ and mutant iga- cells.

The nonencapsulated, IgA protease-positive Haemophilus influenzae strain Rd and serogroup b clinical isolates were found to proliferate in human milk. Growth did not require supplemental X and V factors. In milk, strain Rd synthesized IgA protease, but it was completely inhibited by antibody, so secretory IgA in milk cultures remained intact. Inhibition was largely attributable to IgA1 antibodies. Rd cells also aggregated during growth in milk and showed colony size variation, whereas a protease-negative mutant of Rd (Rd225DK) aggregated less and had uniform colony size. Like differences in protease inhibition, these differences in growth pattern were mediated by secretory IgA1. Thus, milk antibody not only inhibited the extracellular protease but also interacted directly with the enzyme precursor or related antigens on growing bacterial cells. This self-protective property of milk secretory IgA may be an important immunologic attribute for the upper respiratory mucosa of the infant.

Culture Media↗
PubMed1991

Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function.

Dipeptidyl peptidase IV (DP-IV; dipeptidyl-peptide hydrolase, EC 3.4.14.5) is a serine protease with a specificity for cleaving Xaa-Pro dipeptides from polypeptides and proteins. It is found in a variety of mammalian cells and tissues, including those of lymphoid origin where it is found specifically on the surface of CD4+ T cells. Although the functional significance of this enzyme has not been established, a role in T-cell activation and immune regulation has been proposed. Here we report that Ala-boroPro and Pro-boroPro, where boroPro is the alpha-amino boronic acid analog of proline, are potent and specific inhibitors of DP-IV, having Ki values in the nanomolar range. Blocking the N terminus of Ala-boroPro abolishes the affinity of this inhibitor for DP-IV, while removal of the N-terminal residue, to give boroPro, reduces the affinity for DP-IV by 5 orders of magnitude. The dipeptide boronic acids exhibit slow-binding kinetics, while boroPro does not. We also report here that low concentrations of Pro-boroPro inhibit antigen-induced proliferation and interleukin 2 production in murine T-cell lines but do not inhibit the response of these T cells to the mitogen concanavalin A. These results indicate that DP-IV plays a role in antigen-induced, but not mitogen-induced, activation of T lymphocytes.

Animals↗
PubMed1991

Analysis of the immunoglobulin A protease gene of Streptococcus sanguis.

The amino acid sequence T-P-P-T-P-S-P-S is tandemly duplicated in the heavy chain of human immunoglobulin A1 (IgA1), the major antibody in secretions. The bacterial pathogen Streptococcus sanguis, a precursor to dental caries and a cause of bacterial endocarditis, yields IgA protease that cleaves only the Pro-Thr peptide bond in the left duplication, while the type 2 IgA proteases of the genital pathogen Neisseria gonorrhoeae and the respiratory pathogen Haemophilus influenzae cleave only the P-T bond in the right half. We have sequenced the entire S. sanguis iga gene cloned into Escherichia coli. A segment consisting of 20 amino acids tandemly repeated 10 times, of unknown function, occurs near the amino-terminal end of the enzyme encoded in E. coli. Identification of a predicted zinc-binding region in the S. sanguis enzyme and the demonstration that mutations in this region result in production of a catalytically inactive protein support the idea that the enzyme is a metalloprotease. The N. gonorrhoeae and H. influenzae enzymes were earlier shown to be serine-type proteases, while the Bacteroides melaninogenicus IgA protease was shown to be a cysteine-type enzyme. The streptococcal IgA protease amino acid sequence has no significant homology with either of the two previously determined IgA protease sequences, that of type 2 N. gonorrhoeae and type 1 H. influenzae. The differences in both structure and mechanism among these functionally analogous enzymes underscore their role in the infectious process and offer some prospect of therapeutic intervention.

Amino Acid Sequence↗
PubMed1990

Inhibition of IgA1 proteinases from Neisseria gonorrhoeae and Hemophilus influenzae by peptide prolyl boronic acids.

The alpha-aminoboronic acid analog of proline has been synthesized and incorporated into a number of peptides as the COOH-terminal residue. These peptide prolyl boronic acids are potent inhibitors of both the type 1 and type 2 IgA proteinases from Neisseria gonorrhoeae and Hemophilus influenzae, but not of the functionally similar IgA proteinase from Streptococcus sanguis. The best inhibitors synthesized thus far have Ki values in the nanomolar range (4.0 to 60 nM). These results indicate that the N. gonorrhoeae and the H. influenzae enzymes belong to the serine protease family of proteolytic enzymes while that from S. sanguis does not. As a group, the IgA proteinases have been noted for their remarkable specificity; thus, the peptide prolyl boronic acids reported here are the first small synthetic molecules to exhibit a relatively high affinity for the active site of an IgA proteinase and are therefore the first to yield some insight into the active site structure and specificity requirements of these enzymes.

Amino Acid Sequence↗
PubMed1990

Localization of the cleavage site specificity determinant of Haemophilus influenzae immunoglobulin A1 protease genes.

Immunoglobulin A1 (IgA1) proteases are produced by a number of different species of bacteria which cause infection at human mucosal surfaces. The sole substrate of these proteases is human IgA1. Cleavage is within the hinge region of IgA1, although there is variability in the exact peptide bond within the hinge region that is cut by a particular protease. The cleavage site of the Haemophilus influenzae type 1 protease is located four amino acids from the cleavage site of the type 2 enzyme. In this study, the region of the H. influenzae IgA1 protease gene (iga) that determines the cleavage site specificity was localized through the comparison of the type 1 and type 2 genes and the construction and analysis of type 1-type 2 hybrid genes. The hybrid genes were generated by in vivo and in vitro techniques which facilitated the selection and screening of randomly generated hybrids. The cleavage site determinant was found to be within a 370-base-pair region near the amino-terminal coding region, in one of two large areas of nonhomology between the two types of H. influenzae iga genes. DNA sequence analysis of the cleavage site determinant and surrounding regions did not reveal a simple mechanism whereby one enzyme type could be converted to the other type. Comparison of the type 2 gonococcal IgA1 protease gene to the two Haemophilus genes revealed a significant amount of homology around the cleavage site determinant, with the two type 2 genes showing greater homology.

Animals↗
PubMed1989

Tetrapeptide inhibitors of the IgA1 proteinases from type I Neisseria gonorrhoeae.

The six series of unique tetrapeptides and their blocked N-acetyl, C-amide, and N-acetyl-C-amide analogues which comprise the hinge region of human IgA1 (Ser224 to Ser240) have been synthesized and tested as inhibitors of the type 1 IgA1 proteinase elaborated by Neisseria gonorrhoeae (EC 3.4.24.13). Most series had at least one member with an IC50 value less than 1 mM. The most effective inhibitors came from the series Ser-Thr-Pro-Pro (P4-P1) and Pro-Thr-Pro-Ser (P1-P3'). One member from each series had an IC50 value in the low microM range. Magnetic resonance studies (Siemion, I. Z.; et al. Biophys. Chem. 1988, 31, 35) indicate that the various tetrapeptide series appear to have different preferred solution conformations. However, these do not appear to be correlated with affinity for the neisserial proteinase. The most effective inhibitors tend to have a threonine residue adjacent to the N-terminus and the P1 or P1' residues at either the N- or the C-terminus. These relationships are not exclusive however, as other inhibitors, which do not meet these criteria, bind reasonably well. The most effective substrate analogues outlined here are about one-half the size and bind to the neisserial proteinase 2 orders of magnitude more tightly than previously reported inhibitors.

Amino Acid Sequence↗
PubMed1978

IgA protease production as a characteristic distinguishing pathogenic from harmless neisseriaceae.

IgA proteases are extracellular enzymes of bacteria that have human immunoglobulin A of the IgA1 subclass as their only known substrate. The identification of this enzyme in neisseria prompted us to determine whether IgA protease production correlates with pathogenicity within this genus. Multiple clinical isolates of Neisseria gonorrhoeae, N. meningitidis and eight species of non-pathogenic neisseria that commonly colonize the normal human nasopharynx were examined for IgA protease activity. All N. gonorrhoeae and N. meningitidis strains were enzyme positive; all non-pathogenic strains were negative. Among meningococci, the enzyme occurred in strains carried harmlessly in the nasopharynx as well as those isolated from systemic infections. Because mucosal immune defense is largely mediated by antibodies of the IgA isotype, the finding that IgA protease activity is linked specifically to the pathogenic neisseria suggests that the enzyme may be involved in the pathogenesis of neisserial infection.

Gonorrhea↗
PubMed1978

Assay and properties of IgA protease of Streptococcus sanguis.

An assay procedure for streptococcal IgA protease is described which uses isotopically labelled human serum IgA as substrate. Enzyme activity was monitored by the radioactive counts in the Fab alpha product, which was separated from other components in the digestion mixture by electrophoresis. Cleavage of IgA was linear with respect to time using catalytic amounts of the enzyme. Km was calculated to be 5.5 X 10(-6)M, pH optimum 6.0-7.0 at 37 degrees C, and the enzyme was fully inactivated at low concentrations of the metal chelator ethylenediaminetetraacetic acid.

Edetic Acid↗
PubMed1978

Hepatic failure and death from erythropoietic protoporphyria.

A 60-year-old white male presenting with a clinical picture of obstructive jaundice was subsequently found to have erythropoietic protoporphyria. The diagnosis was suspected because of a history of life-long photosensitivity and was confirmed by finding high levels of erythrocyte protoporphyrin. Liver biopsy revealed birefringent deposits of protoporphyrin by polarization microscopy accompanied by severe hepatic injury and fibrosis. The patient died rapidly from liver failure, and at autopsy the biliary tree was patent. Despite the autosomal dominant transmission of erythropoietic protoporphyria, we failed to detect any family members with the disease. This report is concluded with a brief discussion of the liver involvement in erythropoietic protoporphyria.

Cholestasis↗
PubMed1977

Loss of antibody activity in human immunoglobulin A exposed extracellular immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis.

Immunoglobulin A (IgA) proteases are extracellular enzymes elaborated by Neisseria gonorrhoeae, N. meningitidis, and Streptococcus sanguis. These enzymes each cleave human IgA1 at a critically situated prolyl-threonyl peptide bond to yield Fab alpha and Fc alpha fragments. To study their effect on the antibody activity of human IgA, we enzymatically digested a group of five human IgA monoclonal immunoglobulins with high-titer rheumatoid factor or cold agglutinin activity and human serum macroamylase, an amylase-IgA complex. In contrast to four control IgM rheumatoid factor monoclonal proteins, whose activity was unaffected by enzyme, gonococcal and streptococcal IgA proteases caused prompt, major reductions of IgA antibody activity to negligible levels and converted macroamylase activity to amylase of normal size, as determined by molecular sieve chromatography. In addition, both enzymes promptly deagglutinated sensitized cells that had been aggregated by IgA rheumatoid factors, indicating that IgA bound to antigen is also susceptible to enzyme cleavage. Fab fragments of Iga protein Chr, a rheumatoid factor, showed essentially no antigen-binding activity despite the high titers observed with the parent protein. These studies emphasize the high degree of specificity of the microbial proteases for IgA and their potential for interfering with antibody activity in the IgA1 subclass.

Agglutination↗
PubMed1975

Evaluation of human oral organisms and pathogenic Streptococcus for production of IgA protease.

IgA protease is a proteolytic enzyme found in whole human saliva and in dental plaque that cleaves both secretory and myeloma IgA of human origin to yield intact Fabalpha and Fcalpha fragments. To determine which bacteria are capable of producing this enzyme, we have examined a variety of strains normally found in the human oral cavity and a number of streptococci of known Lancefield group serotype. Streptococci of groups A, B, C, D, F, G, H, M, and N, Streptococcus mutans, Streptococcus sanguis, Streptococcus mitior, Streptococcus salivarius, Streptococcus faecalis, Veillonella, Lactobacillus, Actinomyces, Propionibacterium, Bacteroides, and Fusobacterium were grown in liquid medium, and fluids were examined for IgA protease activity. Only S. sanguis and clinically isolated group H streptococci elaborated IgA protease under the culture conditions used. Negative strains could not be stimulated to produce the enzyme when cultured in the presence of secretory IgA. Among the natural oral bacteria, capacity to produce IgA protease is restricted to certain species of Streptococcus, notably those of the group H serotype. Since secretory immunity is mediated by the IgA class of antibody, the presence of this enzyme at mucosal surfaces could modify the secretory immune function.

Actinomyces↗