Cloning of a lymphocyte homing receptor reveals a lectin domain

Lymphocytes express cell surface molecules, termed homing receptors, that mediate their selective attachment to specialized high endothelial venules found within secondary lymphoid organs. Previous work has demonstrated that the adhesive interaction between lymphocytes and the endothelium of peripheral lymph nodes appears to involve a lectin-like activity. Moreover, MEL-14, a monoclonal antibody that blocks lymphocyte-peripheral lymph node binding and presumably recognizes the homing receptor mediating this adhesive interaction, appeared to detect the lectin-like receptor. In this paper we describe the cloning of a murine cDNA that encodes the antigen recognized by the MEL-14 antibody. Characterization of the cDNA encoding the putative mouse peripheral lymph node-specific homing receptor shows that it contains a lectin domain that appears to be involved in the binding of lymphocytes to peripheral lymph node endothelium, thus defining a new type of cellular adhesion molecule. This result supports a novel mechanism for the distribution of lymphocyte populations to various lymphoid organs.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

Further characterization of the interaction between L-selectin and its endothelial ligands.

L-Selectin is a lectin-like receptor on lymphocytes which mediates their attachment to high endothelial venules (HEV) within lymph nodes. Previous work has identified HEV-associated endothelial ligands for L-selectin as sialylated, fucosylated and sulphated glycoproteins of approximately 50 kDa and approximately 90 kDa (Sgp50 and Sgp90). The interaction of L-selectin with these ligands is carbohydrate directed, reflecting the involvement of its amino-terminal, calcium-type lectin domain. It has been reported, and we have confirmed, that anti-Ly22 blocks the adhesive function of L-selectin without reducing its binding to a carbohydrate- based ligand PPME (phosphomannan monoester core from Hansenula hostii). The epitope for this monoclonal antibody depends on the epidermal growth factor (EGF) domain of L-selectin. We demonstrate that anti-Ly22 inhibits the interaction of L-selectin with both of the Sgps, thus establishing that the interaction of L-selectin with HEV can be accounted for by the Sgps. Furthermore, the interaction of trypsin fragments of Sgp50 with L-selectin is inhibitable both by an antibody that maps to the lectin domain and by anti-Ly22. These findings raise the possibility that anti-Ly22 is affecting the function of the lectin domain of L-selectin rather than directly antagonizing the EGF domain. Toward a further characterization of L-selectin's carbohydrate specificity, we show that Sgp50 is partially inactivated by the linkage-specific Newcastle Disease virus sialidase (alpha 2,3 linkage). We additionally demonstrate that a sialyl Lewis x-related tetrasaccharide can interact with L-selectin, as has also been demonstrated for E-selectin and P-selectin.     

Human cytotoxic T cell clones directed against herpes simplex virus-infected cells. IV. Recognition and activation by cloned glycoproteins gB and gD

Results of studies in mice and clinical observations in man indicate that T cell-mediated immunity is important in resistance to herpes simplex virus (HSV) infections. This study was undertaken to elucidate the viral antigen specificity of human HSV-immune T cells. Purified HSV-1 glycoproteins gB-1 and gD-1, cloned and expressed in mammalian cells, were found to stimulate proliferation of, and interleukin 2 (IL 2) production by, peripheral blood lymphocytes (PBL) of HSV seropositive individuals, indicating the presence of memory T cells to gB-1 and gD-1 in individuals with serologic evidence of immunity to HSV. Second, T cell clones, generated by stimulation of PBL with HSV-1, were found to recognize gB-1 or gD-1, as evidenced by the ability of the clones to proliferate in response to stimulation with gB-1 or gD-1 in the absence of exogenous IL 2. Third, HSV-specific T cell clones, lytic for HSV-1 or both HSV-1- and HSV-2-infected autologous target cells, were generated after stimulation of PBL with purified cloned gB-1 or gD-1. Our findings, that human HSV-specific T cells can recognize and be activated by HSV subunit antigens gB-1 or gD-1, imply that these glycoproteins play a role in human T cell-mediated immunity to HSV and support the contention that a gB-1 or gD-1 subunit vaccine may be protective in man.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

The Mel 14 antibody binds to the lectin domain of the murine peripheral lymph node homing receptor

Murine and human leukocytes express surface glycoproteins, termed homing receptors (HRs), containing lectin-like, EGF-like (egf), and complement binding-like domains, that apparently endow these cells with the ability to home to peripheral lymph nodes (pln's) by virtue of an adhesive interaction with the pln postcapillary venule endothelium. The murine pln HR was initially characterized with a rat monoclonal antibody, Mel 14, that was specific for the murine form of the receptor. This work demonstrated that Mel 14 blocked the binding of murine lymphocytes to pln endothelium both in vitro and in vivo, a result consistent with the possibility that this monoclonal antibody recognizes a region of the HR that is involved with endothelium recognition and adhesion. In addition, this antibody also blocked the binding to the HR of PPME, a polyphosphomannan carbohydrate known to inhibit lymphocyte-pln endothelium interactions, suggesting that Mel 14 may recognize the lectin domain of the pln HR. Here we show that, while Mel 14 recognized truncated HR containing both the lectin and egf domains, antibody recognition was lost when the lectin domain alone was expressed. Chimeric molecules, in which regions of the lectin domain of the non-Mel 14-reactive human pln HR were replaced with homologous regions of the murine pln HR, demonstrated that the Mel 14 recognition site is within the NH2-terminal 53 amino acids of the lectin domain. These results suggest that the Mel 14 monoclonal antibody recognizes a determinant within the lectin domain of the pln HR whose conformation may be dependent upon the presence of the egf domain. Since Mel 14 efficiently blocks lymphocyte-endothelial interactions, these results support the hypothesis that the pln HR lectin domain may be directly involved with binding of lymphocytes to a carbohydrate ligand on the pln postcapillary venule endothelium.     

Characterization of a human homologue of the murine peripheral lymph node homing receptor

Lymphocyte trafficking is a fundamental aspect of the immune system that allows B and T lymphocytes with diverse antigen recognition specificities to be exposed to various antigenic stimuli in spatially distinct regions of an organism. A lymphocyte adhesion molecule that is involved with this trafficking phenomenon has been termed the homing receptor. Previous work (Lasky, L., T. Yednock, M. Singer, D. Dowbenko, C. Fennie, H. Rodriguez, T. Nguyen, S. Stachel, and S. Rosen. 1989. Cell. 56:1045-1055) has characterized a cDNA clone encoding a murine homing receptor that is involved in trafficking of lymphocytes to peripheral lymph nodes. This molecule was found to contain a number of protein motifs, the most intriguing of which was a carbohydrate binding domain, or lectin, that is apparently involved in the adhesive interaction between murine lymphocytes and peripheral lymph node endothelium. In this study, we have used the murine cDNA clone to isolate a human homologue of this peripheral lymph node-specific adhesion molecule. The human receptor was found to be highly homologous to the murine receptor in overall sequence, but showed no sequence similarity to another surface protein that may be involved with human lymphocyte homing, the Hermes glycoprotein. The extracellular region of the human receptor contained an NH2 terminally located carbohydrate binding domain followed by an EGF-like domain and a domain containing two repeats of a complement binding motif. Transient cell transfection assays using the human receptor cDNA showed that it encoded a surface glycoprotein that cross reacted with a polyclonal antibody directed against the murine peripheral lymph node homing receptor. Interestingly, the human receptor showed a high degree of sequence homology to another human cell adhesion glycoprotein, the endothelial cell adhesion molecule ELAM.