Comparison of mouse Ly5a and Ly5b leucocyte common antigen alleles.

The family of leucocyte common antigen (LCA) transmembrane glycoproteins is expressed in most hematopoietic cells. Molecular isoforms of the LCA molecule are generated by alternative splicing of a single gene encoded on the murine chromosome 1. Three LCA alleles with different antigenic reactivities have been identified in inbred mouse strains. To investigate the divergence between alleles, cDNA clones to the SJA (Ly5a) LCA gene have been isolated and sequenced. A comparison of this information to the Ly5b allele sequence identifies 12 allele-specific nucleotide changes. These base substitutions correspond to five amino-acid changes within the extracellular domain of the LCA molecule. These amino-acid differences are clustered in a region that also contains the greatest divergence between mouse and rat LCA sequences. Thus, these two mouse LCA alleles exhibit a pattern of sequence conservation that mimics that found over a much broader scale of evolution. Analysis of antigenicity profiles for each of the allelic sequence changes reveals three molecular domains of altered antigenicity that could account for observed serological differences between the two alleles. Sequence information from the 5' end of the Ly5a LCA gene, generated using polymerase chain-reaction techniques on genomic DNA, reveals eight additional nucleotide differences between the Ly5a and Ly5b alleles.

Multiple sites on IL-8 responsible for binding to alpha and beta IL-8 receptors.

To define the structural features important for IL-8 binding to its two known receptors, mutants of IL-8 and melanoma growth-stimulating activity (MGSA) and chimerae consisting of segments of these two chemokines were constructed and purified from the pGEX 2T Escherichia coli expression vector. IL-8 alpha and beta receptors were expressed stably and individually in 293 kidney epithelial cells and HL60 human leukemia cells. The Kd for IL-8 itself and copy numbers for both receptors in transfected cells were comparable. Competition binding with 125I-labeled IL-8, however, showed large differences for several of the IL-8 mutants between alpha and beta receptors. The amino-terminal ELR sequence was important for IL-8 binding to the alpha receptor, but not sufficient for high affinity binding. Both rabbit IL-8 and MGSA share the ELR sequence with human IL-8, but compete poorly with it. The carboxyl terminus distal to amino acid 50 does not seem to mediate high affinity binding to the alpha receptor. A rabbit IL-8/human IL-8 chimera that differs in only eight amino acids from the human IL-8 sequence, was 150-fold lower in its affinity for the alpha receptor than human IL-8. In contrast, both the amino and carboxyl termini appear to be important for binding to the beta receptor. If the ELR sequence of IL-8 was substituted with alanines or if the carboxyl terminus distal to C50 was replaced with the MGSA sequence, a reduction occurred in binding competition. If both changes were introduced simultaneously, binding was abolished. Binding of MGSA was completely prevented by replacement of the ELR sequence with alanines. Ca2+ mobilization in HL60 cells transfected with the alpha or beta receptor was used to assess cell stimulation. The various mutant forms of IL-8 induced receptor activity with a pattern of sensitivity parallel to the competition binding affinities, indicating that both receptors are active.

Phase variation of gonococcal protein II: regulation of gene expression by slipped-strand mispairing of a repetitive DNA sequence.

Expression of outer membrane protein II (P.II) of Neisseria gonorrhoeae is subject to reversible phase variation at a rate of 10(-3)-10(-4)/cell/generation. The signal peptide coding regions of P.II genes contain variable numbers of tandem repeats of the sequence CTCTT. Changes in the number of CTCTT units, leading to frameshifting within the gene, are responsible for changes in P.II expression. Phase variation mediated by the CTCTT repeat also occurred in E. coli, as assayed with a P.II-alkaline phosphatase (phoA) gene fusion. Phase variation in both the gonococcus and E. coli was recA-independent, occurred at similar rates, and involved insertions or deletions of one or more repeat units. The characteristics of the phase variation process were consistent with a model in which expression of P.II genes is regulated by slipped-strand mispairing of the DNA in the CTCTT repeat region.

The role of Tyr13 and Lys15 of interleukin-8 in the high affinity interaction with the interleukin-8 receptor type A.

Interleukin-8 (IL-8) has at least two binding regions for both the A and the B type IL-8 receptors. This study defines an important region between Cys7 and Cys50 that, together with the Glu4-Leu5-Arg6 sequence of the NH2 terminus, accounts for the high affinity binding of IL-8 to the IL-8 A receptor on leukocytes. Utilizing rabbit IL-8 that shares 82% sequence identity with human IL-8, but has 200-fold lower binding affinity for the IL-8 A receptor, residues of the human homologue were sequentially exchanged into the rabbit molecule. Replacement of rabbit His13 and Thr15 with Tyr13 and Lys15 of the human molecule converted the low affinity binding of the rabbit IL-8 to the high affinity binding of human IL-8 as shown by both competitive binding and by Ca2+ mobilization. As a corollary, replacement of the Tyr13 and Lys15 of the human IL-8 with His13 and Thr15 of the rabbit IL-8 reduced binding activity of this mutated human IL-8 200-fold. The site of interaction on the IL-8 receptor type A for the Tyr13 and Lys15 sequence was found to be in the NH2-terminal region of this receptor. A structural pattern of the binding between IL-8 and the A type IL-8 receptor is proposed.

Antigenic and structural differences among six proteins II expressed by a single strain of Neisseria gonorrhoeae.

Gonococci express a family of related outer membrane proteins designated protein II (P.II), which undergo both phase and antigenic variation. Six P.II proteins have been identified in strain FA1090. We developed monoclonal antibodies specific for each P.II protein. Using these antibodies as probes, we purified the six different P.II proteins of this strain. Despite the relatedness of the proteins, we could not purify all of them by a single purification scheme. Four P.II proteins were purified by chromatofocusing, and the remaining two proteins were purified by hydrophobic interaction chromatography on phenyl-Sepharose. The N-terminal amino acid sequence of the proteins showed a high degree of sequence conservation. However, there was variability at specific amino acid residues, giving each P.II protein a unique N-terminal amino acid sequence. Thus P.II proteins of one strain differ among themselves not only in antigenic determinants and primary structure, but also in other characteristics affecting their properties in different chromatographic systems.

Characterization of the neisserial lipid-modified azurin bearing the H.8 epitope.

The pathogenic Neisseria have multiple genes encoding proteins that bind monoclonal antibody (MAb) H.8. We previously reported the cloning and sequencing of a meningococcal gene (laz) encoding an H.8 MAb-binding protein with a consensus lipoprotein processing site, an N-terminal domain containing the epitope for H.8 MAb binding, and a C-terminal domain with extensive similarity to the sequences of azurins from other organisms. In the current study, we showed that the product of the cloned gene could be labelled with palmitic acid, that it was subject to globomycin-sensitive processing, and that it was immunologically cross-reactive with azurin from Pseudomonas aeruginosa. All neisserial species tested, both pathogens and commensals, produced a protein recognized by anti-azurin serum. Southern blots with oligonucleotide probes specific for the azurin domain of the gene showed that it was present in a single copy in the chromosome; it was highly conserved in gonococci and meningococci, and less conserved in commensal Neisseria species.

Reversible phase variation of expression of Neisseria meningitidis class 5 outer membrane proteins and their relationship to gonococcal proteins II.

Neisseria meningitidis class 5 proteins are major outer membrane proteins that share many properties with the proteins II (P.II) of Neisseria gonorrhoeae. We generated two bactericidal monoclonal antibodies, each of which bound specifically to one of the two identified class 5 proteins produced by N. meningitidis FAM18. The monoclonal antibodies also bound to class 5 proteins of a limited number of other meningococcal strains. Using the bactericidal activity of the monoclonal antibodies, we demonstrated that expression of both class 5 proteins was subject to reversible phase variation in vitro. The N-terminal amino acid sequence of a purified class 5 protein revealed striking similarity to the N-terminal amino acid sequence of gonococcal P.II proteins. Using a cloned class 5 gene, we identified three potential class 5 gene loci in N. meningitidis FAM18. These class 5 sequences also had homology with gonococcal P.II gene sequences and contained the CTCTT repeat sequence believed to be important in the regulation of gonococcal P.II expression.

Resistance to meningococcemia apparently conferred by anti-H.8 monoclonal antibody is due to contaminating endotoxin and not to specific immunoprotection.

We evaluated the ability of a monoclonal antibody directed against the common H.8 antigen of pathogenic Neisseria sp. to confer passive protection against meningococcal disease in mice. The apparent protection conferred by antibody purified from tissue culture supernatant was actually the result of endotoxin contamination of buffers and tissue culture media. Endotoxin-free anti-H.8 antibody was not protective. The possibility of endotoxin contamination should be considered when evaluating immunity conferred by passively administered antibody in animal models.

The multi-PDZ domain protein MUPP1 is a cytoplasmic ligand for the membrane-spanning proteoglycan NG2.

A yeast two-hybrid screen was employed to identify ligands for the cytoplasmic domain of the NG2 chondroitin sulfate proteoglycan. Two overlapping cDNA clones selected in the screen are identical in sequence to a DNA segment coding for the most amino-terminal of the 13 PDZ domains found in the multi-PDZ-protein MUPP1. Antibodies made against recombinant polypeptides representing these two clones (NIP-2 and NIP-7) are reactive with the same 250-kDa molecule recognized by anti-MUPP1 antibodies, confirming the presence of the NIP-2 and NIP-7 sequences in the MUPP1 protein. NIP-2 and NIP-7 GST fusion proteins effectively recognize NG2 in pull-down assays, demonstrating the ability of these polypeptide segments to interact with the intact proteoglycan. The fusion proteins fail to bind NG2 missing the C-terminal half of the cytoplasmic domain, emphasizing the role of the NG2 C-terminus in the interaction with MUPP1. The existence of an NG2/MUPP1 interaction in situ is demonstrated by the ability of NG2 antibodies to co-immunoprecipitate both NG2 and MUPP1 from detergent extracts of cells expressing the two molecules. MUPP1 may serve as a multivalent scaffold that provides a means of linking NG2 with key structural and/or signaling components in the cytoplasm.

The H8 antigen of pathogenic Neisseriae.

We cloned and sequenced the H8 gene from N. meningitidis FAM18. The predicted amino acid sequence included a consensus lipoprotein signal sequence processing site, consistent with lipid modification that could account for the unusual electrophoretic and solubilization properties of H8. The amino acid sequence was rich in alanine and proline, especially in an imperfectly periodic region near the amino terminus, which encompassed the epitope recognized by available monoclonal antibodies. In a panel of neisserial strains, the presence of DNA homologous to the H8 gene correlated with the expression of an H8 protein. We cloned a gene from N. meningitidis JB515 that was distinct from the H8 gene but encoded a protein also recognized by an anti-H8 monoclonal antibody. Mice were not protected from meningococcemia by passive immunization with such an antibody.

Genetic and biochemical analyses of protein II.

We compared the structure of P.II proteins of gonococcal strain FA1090 by N-terminal sequence analysis of purified proteins and by DNA sequencing of cloned P.II genes. Regulation of P.II gene expression does not involve major DNA rearrangements, but may involve generation of frame-shifts in unexpressed P.II genes. There are probably 8 or 9 P.II genes, each possessing a common leader sequence, in the gonococcal chromosome.

Recombination among protein II genes of Neisseria gonorrhoeae generates new coding sequences and increases structural variability in the protein II family.

Expression of Neisseria gonorrhoeae Protein II (P.II) is subject to phase variation and antigenic variation. The P.II proteins made by one strain possess both unique and conserved antigenic determinants. To study the mechanism of antigenic variation, we cloned several P.II genes, using as probes a panel of monoclonal antibodies (MAbs) specific for unique determinants. The DNA sequences of three P.II genes showed that they shared a conserved framework, with two short hypervariable (HV) regions being responsible for most of the differences among them. We demonstrated that unique epitopes recognized by the MAbs were at least partially encoded by one of the HV regions. Moreover, we found that reassortment of the two HV regions among P.II genes occurs, generating increased structural and antigenic variability in the P.II protein family.