Cell adhesion in the immune system

Cell adhesion molecules have an important role to play in many facets of the immune system. At a recent meeting their role in leukocyte migration, inflammation, cancer metastasis and lymphocyte development was discussed.     

In vitro oligodendrogliotrophic properties of cell adhesion molecules in the immunoglobulin superfamily: myelin-associated glycoprotein and N-CAM

The occurrence of conserved epitopes in the immune system was investigated on the leukocytes of cattle, river buffalo, sheep, camel, swine and humans by indirect immunofluorescence and flow cytometry. The distribution of the most conservative epitopes on leukocyte sub-populations suggests that the expression pattern of the proteins is similar. Western blotting experiments indicate that the recognized antigens are structural homologues.     

Gain-of-function mutations in FcgammaRI of NOD mice: implications for the evolution of the Ig superfamily

It has been postulated that, during evolution of the Ig superfamily, modifications of the function of individual receptors might occur by acquisition of exons and their subsequent modification, though evidence of this is lacking. Here we have analysed the interaction of mouse IgG subclasses with high-affinity FcgammaRI (CD64) which contains three Ig-like domains and is important in innate and adaptive immunity. This analysis has identified a mechanism by which the postulated modification of newly acquired exons provides gains in function. Thus, the most widely distributed FcgammaRI allele in mice (e.g. BALB/c), bound only a single IgG subclass, IgG2a, with high affinity. However, non-obese diabetic (NOD) mice expressed a unique allele that exhibits broader specificity and, in addition to binding IgG2a, FcgammaRI-NOD bound monomeric IgG3 and bound IgG2b with high affinity, an IgG subclass not bound by FcgammaRI of other mouse strains, either as monomer or multivalent immune complexes. Analysis of mutants of FcgammaRI wherein segments of the interdomain junctions were exchanged between FcgammaRI-BALB and FcgammaRI-NOD identified these regions as having major influence in 'gain-of-function' by the NOD form of FcgammaRI. Nucleotide sequence analysis of intron/exon boundaries encoding the interdomain junctions of the FcgammaRI alleles showed these to have arisen by mutation to alter existing or create new mRNA splice donor/acceptor sites, resulting in generation of modified junctions.     

Functional cooperation of beta1-integrins and members of the Ig superfamily in neurite outgrowth induction

Neurite outgrowth is a central aspect of the ontogenetic formation of neural networks and is regulated by distinct groups of cell surface molecules. One protein involved in neurite elongation and fasciculation is the neural Ig superfamily member F11/contactin. We have shown previously that F11 promotes neurite extension of chick tectal neurons by interaction with the tectal receptor NrCAM, a member of the L1 subgroup of the Ig superfamily. By contrast, it does not induce outgrowth of retinal neurons despite the fact that these cells also express NrCAM, suggesting that in retinal cells the F11-NrCAM interaction alone is not sufficient to induce neurite extension. In this report we present a novel image analysis procedure to quantify neurite outgrowth and use it to demonstrate that F11 enhances the fibronectin-induced outgrowth response of embryonic retinal neurons. We reveal that NrCAM is the neuronal receptor mediating the enhanced outgrowth of retinal neurons, whereas the related F11-binding molecule NgCAM is not involved. Furthermore, we provide evidence that a beta1-integrin may represent the fibronectin-dependent receptor that cooperates indirectly with the F11-NrCAM pathway. Our results support the concept of a combinatorial labeling of cells in nervous system histogenesis by different classes of cell surface proteins, in particular by integrins and molecules of the Ig superfamily.     

A cuticular protein from the moulting stages of an insect

A 22 kDa peptide was purified from prepupal cuticles of 5th instar Calpodes ethlius caterpillars. It was absent earlier in the stadium and from the egg and adult, i.e. it is related to cuticle turnover rather than cuticle structure. It was present at larval and metamorphic moults, showing that it is related to moulting not just metamorphosis. The cDNA corresponding to the 22 kDa peptide was isolated by antibody screening of an epidermal cDNA expression library. Hybridization to Calpodes genomic DNA showed that the gene was present as a single copy. The deduced amino acid sequence is not like any of the sequences of cuticular structural proteins that have been published, but has a 47 amino acid sequence similar to bacteriophage T7 N-acetylmuramoyl-L-alanine amidase (34% identical, 51% similar). The amino acid sequence, the timing of expression in development, and the similarity between the substrate of the bacteriophage amidase and components of insect cuticle, all suggest that the 22 kDa protein may have a role in cleaving chitin-peptide bonds as a prerequisite for digestion of the cuticle by chitinases and proteases.     

Gloverin, an antibacterial protein from the immune hemolymph of Hyalophora pupae

Gloverin is an inducible antibacterial insect protein isolated from pupae of the giant silk moth Hyalophora. It is a basic (pI 8.5) protein with a molecular mass of 13.8 kDa, containing a large number of glycine residues (18.5%) but no cysteine, and has an amino acid sequence that reveals no strong degree of identity with any known proteins. Gloverin inhibits the growth of Escherichia coli at a minimal concentration of 1-3 microM, i.e. less than 5% of the concentration of gloverin in the hemolymph of infected pupae. The prime effect of gloverin, following its interaction with lipopolysaccharide (LPS) in the bacterial envelope, is a specific inhibition of the synthesis of vital outer membrane proteins, leading to an increased permeability of the outer membrane. The activity of gloverin is not affected by heating (100 degrees C, 10 min) but is inhibited by Mg2+ and by free LPS. The gloverin molecule will undergo conformational transitions from a monomeric random coil to an alpha-helix upon transfer from an aqueous to a hydrophobic environment, a property likely to be of importance for its interaction with cell-bound LPS. The activity of gloverin is in many respects similar to that of attacin, another antibacterial protein, originally found in Hyalophora [for a review see Boman, H. G., Faye, I., Gudmundsson, G. H., Lee, J.-Y. & Lindholm, D. A. (1991) Eur J. Biochem. 201, 23-31].     

Characterization of the binding properties of protein LG, an immunoglobulin-binding hybrid protein

Protein LG is a 50-kDa hybrid molecule containing four Ig-light-chain-binding domains from protein L of Peptostreptococcus magnus and two IgG-Fe-binding repeats from streptococcal protein G. Here we analyse the binding of protein LG to Ig from several mammalian species. Protein LG was shown to bind human IgG of all subclasses and other Ig classes that carry kappa chains. The binding to human IgG was only marginally influenced by changes in temperature (4-37 degrees C) or salt concentration (0-1.6 M), and was stable over a wide pH range (pH 4-10). Protein LG bound to Ig from 11 of 12 mammalian species, including those of rabbit, mouse and rat. The affinity constants obtained for the interactions between protein LG and polyclonal IgG from rabbit (4.0 x 10(9) M-1), mouse (1.7 x 10(9) M-1) and rat (1.3 x 10(9) M-1) were similar to the value previously reported for the interaction between the hybrid protein and human polyclonal IgG (5.9 x 10(9) M-1). The interaction between protein LG and a mouse IgG mAb was not influenced by the presence of the specific protein antigen, nor was the binding of this antibody to its ligand affected by protein LG. Inhibition experiments demonstrated that the Ig-binding site of one of the fusion partners retained its ligand-binding capacity when the other component was occupied. Protein LG selectively absorbed 85-90% of the total Ig present in human and rabbit sera and 75-80% of the Ig in sera from mouse and rat. Human serum depleted of C1q, factor D and properdin and preabsorbed by protein LG could be used as a source for other complement factors. These data demonstrate that protein LG is a very versatile Ig-binding protein.     

Cloning of BY55, a novel Ig superfamily member expressed on NK cells, CTL, and intestinal intraepithelial lymphocytes

Expression of the BY55 protein has been shown to be tightly associated with NK and CD8+ T lymphocytes with cytolytic effector activity. To determine the function of this protein, we molecularly cloned BY55 cDNA. The cDNA sequence predicts a cysteine-rich, glycosylphosphatidylinositol-anchored protein of 181 amino acids with a single Ig-like domain weakly homologous to killer inhibitory receptors. Reduction and carboxyamidomethylation of immunoprecipitated BY55 gave a band of 27 kDa, whereas reduction alone led to an 80-kDa species, suggesting that BY55 is a tightly disulfide-linked multimer. RNA blot analysis revealed BY55 mRNAs of 1.5 and 1.6 kb whose expression was highly restricted to NK and T cells. BY55 was expressed on the CD56dim, CD16+ subset of NK cells, which have high cytolytic activity, but was not expressed and was not induced on the CD56bright, CD16-subset of NK cells, a subset with high proliferative, but low cytolytic, capacity. In human tissues, BY55 mRNA was expressed only in spleen, PBL, and small intestine (in gut lymphocytes). BY55 was expressed on all intestinal intraepithelial lymphocytes, which were predominantly CD3+TCRalpha/beta+CD4-CD8+CD11b+CD28-CD45RO+C D56-CD101+CD103+ (alphaEbeta7 integrin). In addition, BY55 was expressed on most CD8+CD28- peripheral blood T cells. These phenotypic relationships suggest that CD8+CD28+ precursor CTL may terminally differentiate into CD8+CD28-BY55+ effector CTL and that some of the peripheral blood CD8+CD28- subset may represent recirculation from mucosal epithelial immune sites.     

Structure and stability of an immunoglobulin superfamily domain from twitchin, a muscle protein of the nematode Caenorhabditis elegans

The NMR solution structure of an immunoglobulin superfamily module of twitchin (Ig 18') has been determined and the kinetic and equilibrium folding behaviour characterised. Thirty molecular coordinates were calculated using a hybrid distance geometry-simulated annealing protocol based on 1207 distance and 48 dihedral restraints. The atomic rms distributions about the mean coordinate for the ensemble of structures is 0.55( +/- 0.09) A for backbone atoms and 1.10( +/- 0.08) A for all heavy atoms. The protein has a topology very similar to that of telokin and the titin Ig domains and thus it falls into the I set of the immunoglobulin superfamily. The close agreement between the predicted and observed structures of Ig 18' demonstrates clearly that the I set profile can be applied in the structure prediction of immunoglobulin-like domains of diverse modular proteins. Folding studies reveal that the protein has relatively low thermodynamic stability, deltaG(H2O)U-F = 4.0 kcal mol(-1) at physiological pH. Unfolding studies suggest that the protein has considerable kinetic stability, the half life of the unfolding is greater than 40 minutes in the absence of denaturant.     

Ribonuclease 4, an evolutionarily highly conserved member of the superfamily

The structural and enzymatic properties of RNase 4 are reviewed. This RNase shows a much higher interspecies similarity (approximately 90%) than the other members of the RNase A superfamily. The enzyme is ubiquitous, with the highest amounts present in liver and lung. Its unique uridine specificity results from alterations in and around the pyrimidine-binding site. In particular, the shortened C-terminus and the side chains of Phe-42, Asp-80 and Arg-101 appear to be involved. RNase 4 binds tightly to the intracellular RNase inhibitor, with a Kd of 4 x 10(-15) M.     

Expression of the immunoglobulin superfamily cell adhesion molecule F3 by oligodendrocyte-lineage cells

We have analysed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins by oligodendrocyte-lineage cells. Biosynthetic labeling of mouse oligodendroglial primary cultures and an oligodendroglial precursor cell line demonstrated that these cells synthesise a variety of different GPI-anchored proteins. GPI-anchored proteins were isolated as a bulk preparation from the precursor cell line, and the individual proteins separated by 2D gel electrophoresis and analysed by microsequencing after tryptic digestion of the separated components. One of the most prominent GPI-anchored proteins synthesised by the cell line was identified as the cell adhesion molecule F3, previously thought to be exclusively expressed by neurons. Western blotting and immunoprecipitation with several polyclonal sera confirmed the expression of F3 by oligodendrocyte-lineage cells and demonstrated the presence of F3 in myelin. Double staining with a panel of oligodendrocyte-specific antibodies and anti-F3 antibodies of cerebellar cultures, as well as oligodendrocytes isolated by panning, showed a colocalization of F3 with oligodendrocyte markers. Oligodendrocyte F3 is shown to be susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage, similar to neuronal F3. Northern blots demonstrated that the oligodendroglial F3 mRNA is the same size as the neuronal message; however, no F3 mRNA could be detected in cortical astrocytes and an astrocytic cell line. Thus, in addition to the expression by neurons, the cell-type specificity of F3 expression must be extended to oligodendroglial cells, underscoring the importance of this Ig superfamily member in the nervous system.     

Cell to substratum adhesion is involved in v-Src-induced cellular protein tyrosine phosphorylation: implication for the adhesion-regulated protein tyrosine phosphatase activity

Protein tyrosine phosphorylation accompanies the integrin-mediated cell to substratum adhesion, and is essential for the progression of G1/S phase of the cell-cycle in normal fibroblasts. To examine how cellular protein tyrosine phosphatase (PTPase) activity is involved in regulating the adhesion-dependent protein tyrosine phosphorylation, we employed fibroblast cells bearing an active form of a protein tyrosine kinase (PTK), v-Src. We found that the v-Src induced tyrosine phosphorylation in certain proteins such as tensin, talin, p120, p80/85 (cortactin) and paxillin was greatly reduced when the cell to substratum adhesion was lost. Readhesion of the cells onto fibronectin restored these phosphorylation events, while this was inhibited by the addition of RGD peptide. The kinase activity of the v-Src was unchanged by the loss of cell to substratum adhesion. On the other hand, treatment with a protein tyrosine phosphatase inhibitor vanadate caused much the same increase in the v-Src-mediated cellular tyrosine phosphorylation between cells adhered to the culture environments and cells kept in suspension. These data suggest that PTPase(s) appears to be more critical than the v-Src PTK in determining the cell adhesion-dependent protein tyrosine phosphorylation. Moreover, most of the protein tyrosine phosphorylations that are mediated by the v-Src but still dependent on the cell adhesion were indeed greatly reduced during an anchorage-independent growth of v-Src cells. Thus our data collectively indicate that the v-Src induced high level of tyrosine phosphorylation in certain types of proteins are still under the control of the integrin(s) or the cell adhesion to culture substratum, and most of these adhesion-regulated high levels of tyrosine phosphorylations are not essential for the transformed phenotype.     

Augmentation and suppression of immune responses to an HIV-1 DNA vaccine by plasmid cytokine/Ig administration

The use of cytokines has shown promise as an approach for amplifying vaccine-elicited immune responses, but the application of these immunomodulatory molecules in this setting has not been systematically explored. In this report we investigate the use of protein- and plasmid-based cytokines to augment immune responses elicited by an HIV-1 gp120 plasmid DNA vaccine (pV1J-gp120) in mice. We demonstrate that immune responses elicited by pV1J-gp120 can be either augmented or suppressed by administration of plasmid cytokines. A dicistronic plasmid expressing both gp120 and IL-2 induced a surprisingly weaker gp120-specific immune response than did the monocistronic pV1J-gp120 plasmid. In contrast, systemic delivery of soluble IL-2/Ig fusion protein following pV1J-gp120 vaccination significantly amplified the gp120-specific immune response as measured by Ab, proliferative, and CTL levels. Administration of plasmid IL-2/Ig had different effects on the DNA vaccine-elicited immune response that depended on the temporal relationship between Ag and cytokine delivery. Injection of plasmid IL-2/Ig either before or coincident with pV1J-gp120 suppressed the gp120-specific immune response, whereas injection of plasmid IL-2/Ig after pV1J-gp120 amplified this immune response. To maximize immune responses elicited by a DNA vaccine, therefore, it appears that the immune system should first be primed with a specific Ag and then amplified with cytokines. The data also show that IL-2/Ig is more effective than native IL-2 as a DNA vaccine adjuvant.     

Oligomerization properties of ERp29, an endoplasmic reticulum stress protein

Leber's congenital amaurosis (LCA) is the earliest and most severe of all inherited retinal dystrophies. Recently, we mapped an LCA gene to chromosome 17p13.1 (LCA1) and ascribed the disease to mutations of the retinal guanylate cyclase (ret GC) gene in a subset of families of North African ancestry. Owing to the genetic heterogeneity of LCA and considering that LCA1 results from an impaired production of cGMP in the retina (with permanent closure of cGMP-gated cation channels), we hypothesized that the activation of the cGMP phosphodiesterase (PDE) could trigger the disease by lowering the intracellular cGMP level in the retina. The rod and cone cGMP-PDE inhibitory subunits were regarded therefore as candidate genes in LCA. Here, we report the exclusion of five rod and cone cGMP-PDE subunits in LCA families unlinked to chromosome 17p13.     

Bacterial expression of tenecin 3, an insect antifungal protein isolated from Tenebrio molitor, and its efficient purification

Tenecin 3, an antifungal protein isolated from the insect Tenebrio molitor larvae, inhibits growth of the fungus Candida albicans. However, the antifungal mechanism and functions of tenecin 3 have not yet been studied due to its very low availability from the natural source. Here we report an expression system of the recombinant tenecin 3 in E. coli, whose amino acid composition is the same with that of the natural tenecin 3. We also devised a simple and easy procedure to isolate the recombinant protein from the bacterial cell extracts. The recombinant tenecin 3 showed an antifungal activity against C. albicans as the natural tenecin 3 did. Therefore large quantities of tenecin 3 can be easily obtained by the expression and purification system of tenecin 3 described in this report.     

In vivo properties of an IgG3-IL-2 fusion protein. A general strategy for immune potentiation

In this report, we describe a strategy for enhancing the immunogenicity of a wide variety of Ags by linking them to IL-2 via an IgG3-IL-2 fusion protein with high affinity for a convenient hapten Ag, dansyl (DNS; N,N-dimethyl-1-aminonaphthalene-5-sulfonyl chloride). This fusion protein, anti-DNS-IgG3-IL-2, combines the functional characteristics of its constituents and has pharmacokinetic properties that are greatly improved over those of IL-2 and a previously described IgG1-IL-2 fusion. The molecule is intact and recoverable from the blood of mice hours after i.p. injection and reaches distant organs throughout the animal. The 7-h in vivo half-life of this molecule is much longer than that of IL-2, addressing a major obstacle in the application of IL-2 to human diseases, including cancer and AIDS. Additionally, the Ab's specificity for the hapten dansyl and the convenient chemistry of dansyl provide a means to link IL-2 to virtually any molecule of interest without the complexities and uncertainties of making IL-2 fusions with each molecule individually. Using hapten-conjugated-BSA (DNS-BSA) as a model Ag we show that the Ab response elicited by anti-DNS-IgG3-IL-2-bound DNS-BSA-Sepharose injected into mice is increased over that of DNS-BSA-Sepharose or anti-DNS-IgG3-bound DNS-BSA-Sepharose. Anti-DNS-IgG3-IL-2 also increased the Ab response to soluble DNS-BSA after a booster injection. This system should be useful in testing the ability of IL-2 to potentiate the immune response to Ag and in screening a large number of potential Ags for use in vaccines. The dramatically improved pharmacokinetics should also overcome one of the major difficulties in applying IL-2 to the treatment of human disease, its short half-life.     

Evolution of the serum amyloid A (SAA) protein superfamily

The serum amyloid A (SAA) superfamily comprises a number of genes and proteins characterized from a range of mammalian species. The majority of members described to date are dramatically induced during the acute-phase response, suggesting an important short-term beneficial role in the response to tissue injury and inflammation. However, important disease associations have also been proposed for certain SAAs during chronic inflammation. The nomenclature of many of the superfamily members has been the result of comparisons with previously reported sequences implying disease association and/or functional relatedness between such members. The evolutionary relationships of the SAA superfamily members have been investigated by comparisons at both the amino acid and the nucleotide level. The results indicate that all members of the superfamily within a species have been undergoing concerted evolution. This has important implications in ascribing functions and disease associations to individual SAA superfamily members and indicates that designations should not be based on the extent of amino acid identity alone but should be made only following direct experimental observation of the proteins themselves.