Protein I: structure, function, and genetics.
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Bacterial Outer Membrane Proteins↗
Biomedical subjects
R C Judd
Publications and source records attributed to R C Judd.
Identification and characterization of peptidoglycan-associated proteins in Neisseria gonorrhoeae.
The principal proteins associated with Neisseria gonorrhoeae peptidoglycan (PG), as identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, are the following: two proteins at approximately 90 kilodaltons (kDa), single major species at both 60 and 44 kDa, a 34- to 36-kDa protein, and three proteins between 28 and 32 kDa. A protein analogous to Escherichia coli Braun lipoprotein was not detected with gonococcal cell wall preparations. The identity of the PG-associated proteins was confirmed immunologically with antibody generated against purified cell walls. Two types of protein species, dithiothreitol extractable (the majority) and alkylation dependent (primarily the 34- to 36-kDa protein), appeared to be associated with the N. gonorrhoeae cell wall fraction. It was found that a crucial step in the extraction of the proteins from the PG fraction was the inclusion of an acetone-water wash of the purified PG pellet. Studies with cell wall preparations obtained from N. gonorrhoeae intrinsically labeled with 32P revealed that the acetone wash was removing phospholipid from the cell wall fraction and thus facilitating protein extraction. Autoradiographic analysis with PG material derived from 125I-surface-labeled cells indicated that the 44-kDa protein is exposed on the surface of the organism even when associated with the PG layer. Radioimmunoprecipitation with anti-PG antibody confirmed these findings. Lectin analysis (wheat germ agglutinin conjugated to horseradish peroxidase) suggested that the 34- to 36-kDa protein is covalently attached to the PG layer.
Acetone↗
Export and intercellular transfer of DNA via membrane blebs of Neisseria gonorrhoeae.
Naturally elaborated membrane bleb material is frequently observed in cultures of Neisseria gonorrhoeae. This material was purified and analyzed for protein, lipopolysaccharide, and nucleic acid content. The electrophoretic protein profiles of two bleb-rich fractions, called BI and BII, were distinct, with only BII containing lipopolysaccharide and outer membrane proteins I and III. Both fractions contained RNA, circular DNA, and linear DNA. Exogenous pancreatic DNase I appeared to hydrolyze all bleb-associated DNA in fraction BI and the linear DNA in fraction BII. The circular DNA molecules associated with fraction BII resisted digestion. Electron microscopy of the bleb fractions verified their DNA content. Fixing blebs with glutaraldehyde before mounting them for microscopy prevented release of internal DNA. Such fixation produced little change in the micrographs of BI; however, only traces of DNA were observed in fixed BII preparations. Incubation of wild-type gonococci in mixtures of DNase and blebs purified from antibiotic-resistant strains resulted in efficient exchange of penicillinase-specifying R plasmids. Recipients incorporated plasmids independently of endogenous and exogenous chromosomal streptomycin resistance markers. These in vitro results suggest that bleb formation by N. gonorrhoeae may serve to transfer plasmids intercellularly in vivo, perhaps constituting a previously unexplored genetic exchange mechanism in these bacteria.
Bacterial Outer Membrane Proteins↗
Purification of outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae.
A system of protein purification, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting, that results in purified outer membrane proteins of the gram-negative bacterium Neisseria gonorrhoeae is described. The proteins, which ranged in apparent molecular mass from approximately 31,000 to approximately 92,000 Da, were located by naphthol blue black staining, eluted from nitrocellulose membranes using 88% formic acid, and precipitated by the addition of concentrated ammonium hydroxide. Up to 65% of the original protein present was recovered by this procedure. The resultant purified protein could then be resuspended in aqueous buffer by brief sonication, making it available for further structural and in vivo immunological analyses. Proteins purified in this manner retain their original antigenicity when probed with polyclonal and monoclonal antibodies, and are structurally unaltered by the purification process. This procedure makes it possible to acquire easily usable quantities of highly insoluble outer membrane proteins of gram-negative bacteria.
Bacterial Outer Membrane Proteins↗
Radioiodination and 125I-labeled peptide mapping of proteins on nitrocellulose membranes.
A rapid procedure for generating dozens of 125I-labeled peptide maps from a protein band excised from a single lane of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel has been developed. Proteins, which can be rapidly purified by 2 X SDS-PAGE separation, are electroblotted onto nitrocellulose paper (NCP) and located by aqueous naphthol blue-black staining. All subsequent steps of radioiodination, and enzyme or chemical cleavage, are carried out on the NCP making it possible to test a variety of cleavage reagents on the same protein sample. The resultant peptidic residues, which can be separated by thin-layer electrophoresis-thin-layer chromatography (2D TLE-TLC), SDS-PAGE, or HPLC, can be used in comparative studies or they can be recovered for further structural and immunological analyses.
Bacterial Proteins↗
Analysis of Biomphalaria glabrata (Gastopoda) hemolymph by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-performance liquid chromatography, and immunoblotting.
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Animals↗
Characterization of protein I from serum-sensitive and serum-resistant transformants of Neisseria gonorrhoeae.
The protein IAs of serum-sensitive (FA635) and serum-resistant (FA638) transformants of Neisseria gonorrhoeae, which have identical pedigrees, have been shown to be different by the use of a monoclonal antibody and were also shown to be different by proteinase K cleavage and primary structural and surface peptide mapping. The difference in structure is within the surface-exposed region of the molecule. The only other difference observed between the two strains was a very slight difference in lipooligosaccharide silver staining in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that protein I alone or in combination with lipooligosaccharide may significantly contribute to serum resistance.
Antigens, Bacterial↗
Evidence for N-terminal exposure of the protein IA subclass of Neisseria gonorrhoeae protein I.
The JS3 and FA638 strains of Neisseria gonorrhoeae bear a protein IA subclass of protein I (P.I). The purified P.Is of surface-labeled strains JS3 and FA638 were cleaved with the N-terminal degradation enzyme leucine amino peptidase (LAP), and the resultant fragments were separated in sodium dodecyl sulfate-polyacrylamide gels. Autoradiography demonstrated that the surface radiolabel was absent in a LAP-generated P.I peptide that was about 1,900 daltons lower in apparent molecular mass than the native P.I in both strains. Moreover, the 4G5 monoclonal epitope, known to be located on the surface of the organism, was also absent in the LAP-generated P.I peptide that was about 1,900 daltons less in apparent molecular mass than the original P.I of strain FA638. These data strongly suggest that the N terminus of the P.IA subclass is exposed on the surface of the bacterium and that this region represents about 5%, or 15 to 20 amino acids, of the total protein.
Antibodies↗
Structure and surface exposure of protein IIs of Neisseria gonorrhoeae JS3.
Colonies of Neisseria gonorrhoeae JS3, each bearing a predominate protein II (PII) type, were derived from a progenitor transparent colony. Five distinct PIIs were identified and isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The PII bands were excised from gels of unlabeled whole cells and from gels containing lysates of surface-radioiodinated bacteria. These were subjected to alpha-chymotrypsin digestion and two-dimensional peptide mapping, which allowed for a comparison of both the primary structures of the PIIs and the identification of surface-exposed regions of the molecules. The results demonstrated that PIIs are unrelated to either Protein I or Protein III in structure but are closely related to one another, sharing about two-thirds of the peptides generated by alpha-chymotrypsin. The remaining third of the peptides varied with each PII, resulting in unique portions of the molecule being exposed on the bacterial surface. However, the variable peptides were not always among the exposed peptides, suggesting that the structural differences in the PIIs occur at a discrete site (or sites) of the PII molecule and not randomly throughout the protein. Such alterations can result in the exposure of distant, nonvariant portions of the molecule to the surface, perhaps by conformational changes. These bacteria can thus present a variety of new immunodeterminant sites to the host during the course of disease.
Amino Acid Sequence↗
Effect of proteolytic enzymes, storage and reduction on the structure and biological activity of pertussigen, a toxin from Bordetella pertussis.
Pertussigen (Ptx), referred to by many different names, including pertussis toxin, was separated into five polypeptide subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a discontinuous Tris-glycine buffer system. Under non-reducing conditions, the apparent molecular weights of the polypeptides (mean 10(-3)) were: S1 (26.3), S2 (24.4), S3 (22.7), S4 (12.2), and S5 (11.3). Under reducing conditions, the apparent molecular weights (mean 10(-3)) were: S1 (28.2), S2 (24.8), S3 (24.3), S4 (12.2) and S5 (13.9). The identity of the individual polypeptide subunits was further confirmed by their unique two-dimensional peptide maps. The polypeptides which showed an apparent increase in molecular weight under reducing conditions were those previously found to contain at least two cysteine residues. Reducing conditions also altered the reactivity of S3 and S2 to polyclonal rabbit antibody in electrophoretic transfer (Western) blot analysis. When Ptx was stored in solution at 4 degrees C, S1 and S5 underwent a gradual decrease in apparent molecular weight, as judged by SDS-PAGE. This decrease occurred in three different buffer systems, and was similar to a decrease in apparent molecular weight of S1 and S5 after treatment with the proteolytic enzymes subtilisin or proteinase K. Neither the changes due to storage nor proteolysis affected the activity of Ptx in regard to hemagglutination, lymphocytosis promotion or histamine sensitization. These changes did, however appear to modify the reactivity of S5 in the Western blot. Both the "endogenous" and enzyme-induced changes in S1 and S5 could be stopped by phenylmethanesulfonyl fluoride. These data suggest that S1 and S5 have exposed determinants in the intact Ptx molecule which are readily cleaved by proteases, but have little bearing on the biological activity of the intact molecule. Resistance to inactivation by proteolytic cleavage may help explain the long duration of Ptx activity within in vivo biological systems.
Animals↗
The use of glycogen-induced guinea pig polymorphonuclear leukocytes to detect inhibitors of chemotaxis found in human serum.
Glycogen-induced guinea pig polymorphonuclear leukocytes (PMNs) were employed in experiments designed to establish the optimal conditions for casein-stimulated chemotaxis. Subsequently, it was shown that guinea pig PMNs, in a serum-free medium, were susceptible to inhibition of migration by three distinct types of cell-directed inhibitors of chemotaxis found in normal or diseased human sera. Comparison of inhibition of migration of both guinea pig and human peripheral PMNs showed that guinea pig and human PMNs were equally susceptible to inhibition by sera from trauma victims; this inhibitor had a molecular weight of about 10,000 d. Human PMNs were slightly more susceptible than were guinea pig PMNs to inhibition of migration by a approximately 110,000-d inhibitor found in normal human sera. On the other hand, guinea pig PMNs were somewhat more susceptible to inhibition of migration by a approximately 400,000-d inhibitor of chemotaxis that was analogous to the inhibitor found in anergic serum. This information shows that guinea pig PMNs, in a serum-free medium, may be substituted for human cells in quantitative assays for these human serum factors.
Adult↗
Structural analysis of the variable major proteins of Borrelia hermsii.
Borrelia hermsii undergoes spontaneous antigenic variation in vivo and in vitro. Serotype specificity is associated with expression of one of a family of molecular weight-variable proteins, the pI proteins. We studied the structure of the pI proteins as well as the molecular weight-invariable pII proteins of three serotypes of B. hermsii HS1: C, 7, and 21. The techniques used were one-dimensional (1-D) mapping of Staphylococcus aureus V8 protease-generated peptides and two-dimensional (2-D) mapping of alpha-chymotrypsin-generated peptides. The pI and pII proteins were isolated by excision of polypeptides from stained polyacrylamide gel electropherograms. The 1-D peptide patterns were visualized by fluorography of intrinsically [14C]leucine-labeled proteins or by silver stain. Before 2-D mapping, polypeptides in excised gel fragments were labeled with 125I in the presence of chloramine-T. We also compared the 2-D peptide maps of pI proteins, pI7 and pI21, after their surface-exposed portions were radioiodinated using 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenylglycoluril (Iodogen). The I-D and 2-D peptide maps demonstrated the following: (a) pI proteins of the three serotypes have few V8 protease- or chymotrypsin-generated peptides in common, and (b) pI proteins of each serotype appear to be identical. The findings suggest that pI protein variability derives from extensive differences in the amino acid sequences of these proteins.
Autoradiography↗
125I-peptide mapping of protein III isolated from four strains of Neisseria gonorrhoeae.
Gonococcal outer-membrane protein I (PI) and PIII were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from reduced and unreduced whole-cell and outer-membrane lysates of four strains of nonpiliated (P-), transparent (O-) Neisseria gonorrhoeae. These proteins were radioiodinated and digested with alpha-chymotrypsin. The resultant 125I-peptides were then resolved by high-voltage thin-layer electrophoresis, followed by ascending thin-layer chromatography, and visualized by autoradiography. Results corroborated previous observations regarding the structural relationships of PIs having different apparent subunit molecular weights. All PIIIs had very similar apparent primary structures, regardless of the strain from which they were isolated, the source (i.e., whole cells or outer membranes), or the reduction state of the sodium dodecyl sulfate lysates. By the techniques used, it appeared that PIII is structurally similar in all of the gonococcal strains studied, even though each strain had structurally unique PIs.
Bacterial Outer Membrane Proteins↗
Surface peptide mapping of protein I and protein III of four strains of Neisseria gonorrhoeae.
Whole cells and isolated outer membranes (OMs) of four strains of gonococci were surface radioiodinated with either lactoperoxidase or Iodogen (Pierce Chemical Co., Rockford, Ill.). These preparations were solubilized in sodium dodecyl sulfate and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Surface-radioiodinated protein I (PI) and PIII bands were excised from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and digested with alpha-chymotrypsin, and the resultant 125I-peptide fragments were resolved by high-voltage electrophoresis and thin-layer chromatography (i.e., surface peptide mapping). Radioemitting peptidic fragments were visualized by autoradiography. Results demonstrated that the PI molecule of each gonococcal strain studied had unique iodinatable peptides exposed on the surface of whole cells and OMs, whereas PIIIs appeared to have the same portion of the molecule exposed on the surface of bacteria or OMs, regardless of the gonococcal strain from which they were isolated. Many more radiolabeled peptides were seen in surface peptide maps of PIs from radiolabeled OMs than in those from radioiodinated whole cells, whereas different peptidic fragments were seen in the surface peptide maps of PIIIs from radiolabeled OMs than were seen in those from radiolabeled whole cells. These data suggest that PI may contribute strain-specific antigenic determinants and PIII may contribute cross-reactive determinants and that the surface exposure of PI and PIII is different in isolated OMs than in the OM of intact gonococci.
Bacterial Outer Membrane Proteins↗
Structural analysis of chlamydial major outer membrane proteins.
The primary structure and surface exposure of the major outer membrane protein (MOMP) isolated from 14C intrinsically or 125I extrinsically radiolabeled Chlamydia trachomatis serotypes D/UW-3, G/UW-57, H/UW-4, I/UW-12, and L2/434 and the Chlamydia psittaci meningopneumonitis strain were analyzed by two different peptide-mapping techniques. Radiolabeled proteins were digested with either Staphylococcus aureus V8 protease, the patterns of peptide fragments produced being displayed by sodium dodecyl sulfate gel electrophoresis, or alpha-chymotrypsin, the peptides being analyzed after separation by high-voltage electrophoresis and thin-layer chromatography. The comparative structural data obtained from these two different techniques were remarkably similar. From these data, the following points could be made. (i) MOMPs are structurally heterogeneous between members of chlamydial species; the C. psittaci MOMP was clearly distinct from each of the C. trachomatis MOMPs. (ii) Considerable structural homology occurs among MOMPs from different C. trachomatis serotypes; however, distinct differences in the primary structure of each C. trachomatis MOMP were evident. (iii) These observed differences were most obvious in peptide maps of MOMPs isolated from chlamydiae that had been surface labeled by lactoperoxidase-mediated radioiodination. The surface-exposed portions of the MOMPs from serotypes L2 and D were very similar. In contrast, those from serotypes G, H, and I were quite different. These structural data are in agreement with the serospecificities described for these proteins.
Amino Acid Sequence↗
Gonococcal ribosomes as skin test antigens. I. Preliminary tests of sensitivity and specificity.
Ribosomes isolated from N. gonorrhoeae and N. meningitidis were used as skin test antigens in guinea-pigs which had been previously sensitized with killed Neisseria cells in incomplete Freund adjuvant. Intradermal injection of ribosomes from skin test dose N. gonorrhoeae into the skin of animals sensitized to the homologous organism was characterized by a specificity based upon ribosomal skin test dose 50's (STD50) sensitivity which was at least five-fold greater than that of the animals sensitized to the heterologous organism. The sensitized guinea-pigs had lost their skin-test reactivity within 3 months of the cessation of immunization but regained it immediately on reimmunization. The results of this model system indicate that gonococcal ribosomes may be used as antigens in skin testing or other testing modes in gonorrhoea.
Animals↗
Gonococcal ribosomes as skin test antigens. II. Precision Of the Method, Attempts To Identify The Ribosomal Antigen, and Correlation with The Macrophage Migration Inhibition test.
Gonococcal crude ribosome preparation was found to be specific and sensitive when used to elicit delayed hypersensitivity reactions in sensitized guinea-pigs. Gonococcal crude ribosomes possess the ability to react with humoral antibody in sensitized animals; thus, ribosomes may be of value as an antigen in a sero-diagnostic test for gonorrhoea. Ribosomal protein or RNA, or gonococcal cell components including protoplasm, cell walls, and pilin, were less reactive than intact ribosomes. The ribosome preparation was used in an in vitro correlate of delayed hypersensitivity, suggesting that the human immune response in gonorrhoea might be studied using the crude ribosome in in vitro experiments with human peripheral lymphocytes.
Animals↗