Glycosylation in immune cell trafficking.

Leukocyte recruitment encompasses cell adhesion and activation steps that enable circulating leukocytes to roll, arrest, and firmly adhere on the endothelial surface before they extravasate into distinct tissue locations. This complex sequence of events relies on adhesive interactions between surface structures on leukocytes and endothelial cells and also on signals generated during the cell-cell contacts. Cell surface glycans play a crucial role in leukocyte recruitment. Several glycosyltransferases such as alpha1,3 fucosyltransferases, alpha2,3 sialyltransferases, core 2 N-acetylglucosaminlytransferases, beta1,4 galactosyltransferases, and polypeptide N-acetylgalactosaminyltransferases have been implicated in the generation of functional selectin ligands that mediate leukocyte rolling via binding to selectins. Recent evidence also suggests a role of alpha2,3 sialylated carbohydrate determinants in triggering chemokine-mediated leukocyte arrest and influencing beta1 integrin function. The recent discovery of galectin- and siglec-dependent processes further emphasizes the significant role of glycans for the successful recruitment of leukocytes into tissues. Advancing the knowledge on glycan function into appropriate pathology models is likely to suggest interesting new therapeutic strategies in the treatment of immune- and inflammation-mediated diseases.

PSGL-1 function in immunity and steady state homeostasis.

The substantial importance of P-selectin glycoprotein ligand 1 (PSGL-1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL-1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin-PSGL-1 interaction requires branched O-glycan extensions on specific PSGL-1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O-glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL-1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O-glycans on PSGL-1 and provide the molecular interactions responsible for the velcro-like catch bonds that support leukocyte rolling. Such binding of PSGL-1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL-1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T-cell homing to secondary lymphoid organs. PSGL-1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O-glycan modifications of PSGL-1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL-1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T-cell traffic. The multi-level influence of PSGL-1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O-glycans. However, central as specific O-glycosylation is to PSGL-1 function, in vivo regulation of PSGL-1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL-1 glycosylation and to update understanding of PSGL-1 functional scope.

CD22 and Siglec-G: B-cell inhibitory receptors with distinct functions.

Siglecs (sialic acid-binding immunoglobulin-like lectins) are sialic acid-binding proteins, which are expressed on many cell types of the immune system. B cells express two members of the Siglec family, CD22 (Siglec-2) and Siglec-G, both of which have been shown to inhibit B-cell signaling. CD22 recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 (SHP-1) to immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and inhibits B-cell receptor (BCR)-induced Ca2+ signaling on normal B cells. CD22 interacts specifically with ligands carrying alpha2-6-linked sialic acids. Interaction with these ligands in cis regulates the association of CD22 with the BCR and thereby modulates the inhibitory function of CD22. Interaction of CD22 to ligands in trans can regulate both B-cell migration as well as the BCR signaling threshold. Siglec-G is a recently identified protein with an inhibitory function restricted to a B-cell subset, the B1 cells. Siglec-G inhibits Ca2+ signaling specifically in these cells. In addition, it controls the cellular expansion and antibody secretion of B1 cells. Thus, both Siglecs modulate BCR signaling on different B-cell populations in a mutually exclusive fashion.

Regulation of Notch signaling during T- and B-cell development by O-fucose glycans.

Notch signaling is required for the development of all T cells and marginal zone (MZ) B cells. Specific roles in T- and B-cell differentiation have been identified for different Notch receptors, the canonical Delta-like (Dll) and Jagged (Jag) Notch ligands, and downstream effectors of Notch signaling. Notch receptors and ligands are post-translationally modified by the addition of glycans to extracellular domain epidermal growth factor-like (EGF) repeats. The O-fucose glycans of Notch cell-autonomously modulate Notch-ligand interactions and the strength of Notch signaling. These glycans are initiated by protein O-fucosyltransferase 1 (Pofut1), and elongated by the transfer of N-acetylglucosamine (GlcNAc) to the fucose by beta1,3GlcNAc-transferases termed lunatic, manic, or radical fringe. This review discusses T- and B-cell development from progenitors deficient in O-fucose glycans. The combined data show that Lfng and Mfng regulate T-cell development by enhancing the interactions of Notch1 in T-cell progenitors with Dll4 on thymic epithelial cells. In the spleen, Lfng and Mfng cooperate to modify Notch2 in MZ B progenitors, enhancing their interaction with Dll1 on endothelial cells and regulating MZ B-cell production. Removal of O-fucose affects Notch signaling in myelopoiesis and lymphopoiesis, and the O-fucose glycan in the Notch1 ligand-binding domain is required for optimal T-cell development.

T-cell growth, cell surface organization, and the galectin-glycoprotein lattice.

Basal, activation, and arrest signaling in T cells determines survival, coordinates responses to pathogens, and, when dysregulated, leads to loss of self-tolerance and autoimmunity. At the T-cell surface, transmembrane glycoproteins interact with galectins via their N-glycans, forming a molecular lattice that regulates membrane localization, clustering, and endocytosis of surface receptors. Galectin-T-cell receptor (TCR) binding prevents ligand-independent TCR signaling via Lck by blocking spontaneous clustering and CD4-Lck recruitment to TCR, and in turn F-actin transfer of TCR/CD4-Lck complexes to membrane microdomains. Peptide-major histocompatibility complexes overcome galectin-TCR binding to promote TCR clustering and signaling by Lck at the immune synapse. Galectin also localizes the tyrosine phosphatase CD45 to microdomains and the immune synapse, suppressing basal and activation signaling by Lck. Following activation, membrane turnover increases and galectin binding to cytotoxic T-lymphocyte antigen-4 (CTLA-4) enhances surface expression by inhibiting endocytosis, thereby promoting growth arrest. Galectins bind surface glycoproteins in proportion to the branching and number of N-glycans per protein, the latter an encoded feature of protein sequence. N-glycan branching is conditional to the activity of Golgi N-acetylglucosaminyl transferases I, II, IV and V (Mgat1, 2, 4, and 5) and metabolic supply of their donor substrate UDP-GlcNAc. Genetic and metabolic control of N-glycan branching co-regulate homeostatic set-points for basal, activation, and arrest signaling in T cells and, when disturbed, result in T-cell hyperactivity and autoimmunity.

Unique antigen of cultured Hodgkin's cells. A putative sialyltransferase.

Hodgkin's disease-derived giant cell lines (HD-cells) express high levels of ectosialyltransferase activity presumed to be a galactose-specific lectin recognizing the desialylated 3-fucosyl-N-acetyllactosamine structure (X-hapten). Both the anti-X-hapten monoclonal antibody VIM-D5 and a polyclonal antiserum to another galactose-lectin, the hepatic asialoglycoprotein receptor (HBP), recognize a 55,000-mol wt HD-cell protein (Paietta, E., R. J. Stockert, A. G. Morell, V. Diehl, and P. H. Weirnik. 1986. Proc. Natl. Acad. Sci. USA. 83:3451-3455.) That the expression of the 55,000-mol wt protein is restricted to HD-cells among X-hapten positive cells lines is confirmed in this study. The 55,000-mol wt protein is shown to be present on the cell surface and intracellularly, where an additional immunocrossreactive 150,000-mol wt protein is recognized. Extraction of the 55,000 mol wt protein from HD-cell lysates by affinity chromatography results in the loss of sialyltransferase activity. While evidence for a single protein possessing both the antigenic and the enzymatic activity is not direct, these results suggest that the ectosialyltransferase unique to HD-cells is a 55,000-mol wt membrane glycoprotein possessing the X-hapten oligosaccharide.

Targeting the gram-negative bacteria peptidoglycan synthase MraY as a new approach for monoclonal antibody anti-bacterial activity.

The use of antibiotics to target bacteria is a well-validated approach for controlling infections in animals and humans. Peptidoglycan biosynthesis is a crucial process in bacteria, and the conserved peptidoglycan synthase MraY is an attractive target for drug design. However, due to the lack of detailed MraY structural information, antibiotics targeting MraY have not yet been developed. In the present study, 2 hydrophilic regions of MraY from Escherichia coli were expressed as a fusion protein and used to raise a monoclonal antibody in mice. We confirmed that the MraY amino acid sequence PESHFSKRGTPT forms the core epitope recognized by the monoclonal antibody M-H11. Furthermore, our results show that M-H11 effectively controls Escherichia coli BL21 (DE3) plysS infection, both in vitro and in vivo. Our results may be of great value in the search for novel approaches used to control bacterial infections.

DTDP-rhamnosyl transferase RfbF, is a newfound receptor-related regulatory protein for phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3.

Bacteriophages and their hosts are continuously engaged in evolutionary competition. Here we isolated a lytic phage phiYe-F10 specific for Yersinia enterocolitica serotype O:3. We firstly described the phage receptor was regulated by DTDP-rhamnosyl transferase RfbF, encoded within the rfb cluster that was responsible for the biosynthesis of the O antigens. The deletion of DTDP-rhamnosyl transferase RfbF of wild type O:3 strain caused failure in phiYe-F10 adsorption; however, the mutation strain retained agglutination with O:3 antiserum; and complementation of its mutant converted its sensitivity to phiYe-F10. Therefore, DTDP-rhamnosyl transferase RfbF was responsible for the phage infection but did not affect recognition of Y. enterocolitica O:3 antiserum. Further, the deletions in the putative O-antigen biosynthesis protein precursor and outer membrane protein had no effect on sensitivity to phiYe-F10 infection. However, adsorption of phages onto mutant HNF10-ΔO-antigen took longer time than onto the WT, suggesting that deletion of the putative O-antigen biosynthesis protein precursor reduced the infection efficiency.

Monospecific antiserum to rat spermidine synthase and its application to rat tissues and several mammals.

Monospecific antiserum to rat spermidine synthase was prepared by immunization of rabbits with purified enzyme protein from rat prostate, and its usefulness for analysis of spermidine synthase protein in not only rat tissues but also several other mammals was demonstrated by Western blotting and immunotitration of the enzyme activity. Application of the antiserum for elucidating the relationship between the enzyme activity and protein in normal rat tissues strongly suggested that marked difference in spermidine synthase activity among rat tissues depends solely on the difference in the amount of enzyme protein. Also, application of the antiserum for analyzing spermidine synthase from liver of mouse, rat, guinea pig, pig, and human, showed that the enzymes had a similar subunit molecular weight of 35,000 and a cross-reactivity with the antiserum, exhibiting almost the same immunoreactivity to mouse enzyme as to rat enzyme. Thus, it was suggested that the antiserum would be useful for further studies of mammalian spermidine synthase from the viewpoints of enzymology and molecular biology.

Differences in the immune responses of mice and sheep to an aromatic-dependent mutant of Salmonella typhimurium.

A live mutant aroA Salmonella serotype Typhimurium ovine strain (S25/1) could be cultured from tissues of mice for up to 90 days after oral infection. Following vaccination, high levels of Salmonella-specific serum IgM, IgG and IgA were produced in addition to high levels of specific intestinal IgA. Moreover, there was also evidence of Salmonella-specific cell-mediated immunity in vaccinated mice in the form of strong delayed-type hypersensitivity and the production of interferon-gamma (IFN-tau) by spleen cells stimulated with Salmonella antigen. The aroA strain was also recovered from the mesenteric lymph nodes and most tissues examined from sheep vaccinated by the oral route. Salmonella-specific IgM was detected in the serum; however, specific IgG responses were very low and there was an absence of specific copro-antibody. Although strong Salmonella-specific lymphocyte proliferative responses were detected, they did not result in the production of IFN-tau and flow cytometric analysis revealed that the proliferating cells were predominantly B lymphocytes. Despite the absence of strong vaccine-specific immune responses in vaccinated sheep compared with those seen in mice, both mice and sheep were protected against challenge with virulent wild-type strain S25/1.

Immunity and vaccine development in Pasteurella multocida infections.

The role of LPS in immunity was studied using monoclonal antibodies (MAbs) and active immunisation experiments. A panel of six MAbs produced against Pasteurella multocida serotype B:2 reacted with the LPS of serotypes B:2 and B:5, but not with other serotypes. The MAbs could opsonise P. multocida for phagocytosis by mouse macrophages, but were not bactericidal in the presence of complement. They conferred only partial passive protection in mice. Similar results showing only partial protection were obtained when purified LPS was used to actively immunise mice prior to challenge, suggesting that LPS plays a partial role in immunity to infection. The aroA gene from P. multocida serotypes A:1 and A:3 was cloned and inactivated by insertion of a kanamycin resistance gene. The mutated gene was re-introduced onto the chromosome by allelic exchange. The resultant aroA mutants were highly attenuated in a mouse model system, with a 6-log decrease in ID50. Virulence could be restored by complementation with a functional aroA gene. Mice immunised with two doses of the live mutants were protected against lethal challenge with the homologous parental strain, but not against the heterologous strain. P. multocida A:1 and A:3 expressed unique proteins when grown in iron-restricted medium. Moreover, the outer membrane (OM) fractions of these cells contained novel proteins of 75 kDa, 85 kDa and 94 kDa molecular mass. Mice were immunised with OM fractions prepared from serotype A:3 grown in iron-restricted (OM Fe-) or iron-replete (OM Fe+) media. When low challenge doses were used, both immunogens protected mice against serotype A:3, but only the OM Fe- fraction protected mice against heterologous challenge with serotype A:1. When higher challenge doses were used, only partial protection was observed.

Evaluation of an aroA mutant Salmonella typhimurium vaccine in chickens using modified semisolid Rappaport Vassiliadis medium to monitor faecal shedding.

In groups of chickens vaccinated orally or intramuscularly with a live aroA mutant Salmonella typhimurium vaccine strain and then experimentally inoculated with 10(8) CFU of wild type S. typhimurium or 10(9) CFU of S. enteritidis, faecal shedding of the vaccine and wild type strains was monitored by the buffered peptone water-modified semisolid Rappaport Vassiliadis medium method, which detected less than 10(2) CFU per gram of faeces. The vaccine strain was shed in the faeces for up to 26 days. Vaccination failed to reduce the faecal shedding of wild type S. typhimurium or S. enteritidis. The variation in the shedding patterns of chickens within each group was greater than between treatment groups.

Involvement of lipoprotein PpiA of Streptococcus gordonii in evasion of phagocytosis by macrophages.

Streptococcus gordonii is a commensal gram-positive bacterium that resides in the human oral cavity, and is one of the most common causes of infective endocarditis (IE). Bacterial surface molecules play an important role in establishing IE, and several S. gordonii proteins have been implicated in binding to host cells during the establishment of IE. In this study, we identified a putative lipoprotein, peptidyl-prolyl cis/trans isomerase (PpiA), and clarified its role in evasion of phagocytosis by macrophages. Attenuation of the gene encoding prolipoprotein diacylglyceryl transferase (Lgt) altered the localization of PpiA from the cell surface to the culture supernatant, indicating that PpiA is lipid-anchored in the cell membrane by Lgt. Both human and murine macrophages showed higher phagocytic activity towards ppiA and lgt mutants than the wild-type, indicating that the presence of PpiA suppresses phagocytosis of S. gordonii. Human macrophages treated with dextran sulfate had significantly impaired phagocytosis of S. gordonii, suggesting that class A scavenger receptors in human macrophages are involved in the phagocytosis of S. gordonii. These results provide evidence that S. gordonii lipoprotein PpiA plays an important role in inhibiting phagocytic engulfment and in evasion of the host immune response.

Lipoproteins are critical TLR2 activating toxins in group B streptococcal sepsis.

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.

A monoclonal antibody against a carbohydrate epitope in lipopolysaccharide differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae, and Chlamydia trachomatis.

Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C. pneumoniae, and C. trachomatis in double labeling experiments of infected cell monolayers and by enzyme-linked immunosorbent assay (ELISA). The epitope specificity of mAb S69-4 was determined by binding and inhibition assays using bacteria, LPS, and natural or synthetic Kdo oligosaccharides as free ligands or conjugated to BSA. The mAb bound preferentially Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4)Kdo(2-->4) with a K(d) of 10 microM, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2-->4)Kdo(2-->4) Kdo but not with Kdo(2-->8)Kdo(2-->4)Kdo, Kdo disaccharides in 2-->4- or 2-->8-linkage, or Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin-layer chromatography (TLC) plates in amounts of <10 ng by immunostaining. Due to the high sensitivity achieved in this assay, the antibody also detected in vitro products of cloned Kdo transferases of Chlamydia. The antibody can therefore be used in medical and veterinarian diagnostics, general microbiology, analytical biochemistry, and studies of chlamydial LPS biosynthesis. Further contribution to the general understanding of carbohydrate-binding antibodies was obtained by a comparison of the primary structure of mAb S69-4 to that of mAb S45-18 of which the crystal structure in complex with its ligand has been elucidated recently (Nguyen et al., 2003, Nat. Struct. Biol., 10, 1019-1025).

Preliminary quantification of immunological relationships among the transaldolases of the genus Bifidobacterium.

The immunological relatedness among the transaldolases (dihydroxyacetone transferase, E.C. 2.2.1.2) of twenty species of the genus Bifidobacterum has been tested by the microcomplement fixation method, using B. thermophilum (B. ruminale) RU326 (= ATCC 25866), B. cuniculi RA93 (= ATCC 27916) and B. 'minimum' (DNA homology group) F392 (= ATCC 27916) as references. Based on the serological relationships of the transaldolases, expressed either as indices of dissimilarity or as immunological distances, the twenty species of the genus Bifidobacterium were arranged into clusters. These clusters generally coincided with the immunological groups obtained previously by the immunodiffusion method (Sgorbati and Scardovi, 1979).

Immunological relationships among transaldolases in the genus Bifidobacterium.

Antisera were prepared against electrophoretically homogeneous transaldolase (dihydroxyacetone transferase, E.C. 2.2.1.2.) of Bifidobacterium thermophilum (B. ruminale) RU326 (ATCC25866), B. cuniculi RA93 (ATCC27916) and B. 'minimum' (homology group) F392 (ATCC 27538). Crude extracts of eighty six strains previously assigned to twenty one species of the genus Bifidobacterium on the basis of deoxyribonouclelic acid (DNA) homology (DNA-DNA hybridization), were compared by double diffusion tests on Ouchterlony plates. Eight groups of identical antigenic specificity were recognized. By analysis of the spur formation, the groups of identical specificity were arranged in preliminary sequences of decreasing similarity to each of the three homologous transaldolases used as reference points. The relationships between immunological data and the genetic similarity among the species of the genus measured by means of DNA-DNA hybridization were discussed together with some relevant points of bifidal ecology.