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Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N- and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.

Circulating human natural killer (NK) lymphocytes have been functionally defined by their ability to exert cytotoxic activity against MHC class I-negative target cell lines, including K562. Therefore, it was proposed that NK cells recognized the “missing self.” We show here that the Ig-like CD160 receptor expressed by circulating CD56dim+NK cells or IL-2-deprived NK cell lines is mainly involved in their cytotoxic activity against K562 target cells. Further, we report that HLA-C molecules that are constitutively expressed by K562 trigger NK cell lysis through CD160 receptor engagement. In addition, we demonstrate, with recombinant soluble HLA-Cw3 and CD160 proteins, direct interaction of these molecules. We also find that CD158b inhibitory receptors partially interfere with CD160-mediated cytotoxicity, whereas CD94/CD159a and CD85j have no effect on engagement with their respective ligands. Thus, CD160/HLA-C interaction constitutes a unique pathway to trigger NK cell cytotoxic activity.

Monoclonal antibody MEM-148 was previously shown to recognize CD18 chains in a free form unassociated within leukocyte integrin heterodimers, but yet it is paradoxically able to induce a high-affinity conformation in the native, cell surface expressed LFA-1 molecules.Our results based on kinetics of binding, immunoprecipitation and cell-aggregation experiments demonstrate that the mAb does bind to and stabilizes a specific conformation of LFA-1 heterodimers apparently distinguished by an increased affinity to its cellular ligand(s).A similar high-affinity conformation of LFA-1, in which the MEM-148 epitope becomes exposed, is induced also by a Mg 2+ /EDTA or low pH (5.5-6.5) treatments which may mimic physiologically relevant situations in normal or inflamed tissues.Thus, mAb MEM-148 is a novel valuable tool for detection and induction of specific conformations of human leukocyte imegrins.

The CDw108 glycoprotein is expressed on the surface of some leukemic cell lines, erythrocytes and on activated lymphocytes. Its surface expression is rapidly upregulated following various activating stimuli (PHA, PWM, Con A, PMA, anti-CD3) and subsequently gradually decreases. The molecule is anchored in the membrane via glycosylphosphatidylinositol (GPI) moiety, it has molecular mass of 75-80 kDa and pI of 5.0-5.5. Endoglycosidase F and H reduce its apparent size as determined by SDS PAGE by approx. 15 and 22 kDa, respectively. It is a component of large, detergent-resistant GPI-complexes associated with protein kinases. In addition to the previously described identity of CDw108 with the JMH blood group antigen, we demonstrate here its identity to the previously described glycoprotein recognized by monoclonal antibodies H105 and KS.2, and exclude its identity with another GPI-anchored glycoprotein of similar size, melanotransf errin (gp97).

An unusual CD18 monoclonal antibody (mAb) MEM-148 binds, in contrast to standard CD18 mAbs, specifically to peripheral blood monocytes and neutrophils activated by various stimuli such as phorbol myristate acetate, opsonized zymosan, heat-aggregated immunoglobulin, and (after priming with lipopolysaccharide, tumor necrosis factor, or granulocyte-macrophage colony-stimulating factor) also by formyl-methionyl-leucyl-phenylalanine. In addition, in vivo activated neutrophils obtained from urine of patients following recent prostatectomy were also strongly positive for MEM-148. On the activated myeloid cells the mAb recognized a 65- to 70-kd protein identified immunochemically and by mass spectrometric peptide sequencing as a membrane-anchored fragment of CD18 (the common chain of leukocyte integrins) produced by proteolytic cleavage. The CD18 fragment originated mainly from integrin molecules stored intracellularly in resting cells, it was unassociated with CD11 chains, and its formation was inhibited by several types of protease inhibitors. Thus, the 65- to 70-kd CD18 fragment represents a novel abundant activation marker of myeloid cells of so far unknown function but possibly involved in conformational changes in leukocyte integrin molecules resulting in increased affinity to their ligands.

SIT (SHP2-interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR-mediated recruitment of SH2 domain-containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2-containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR-mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2-binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine-based signaling motif Y(168) ASV completely abrogates the ability of SIT to inhibit T cell activation. Co-precipitation experiments revealed that the tyrosine kinase p50(csk) could represent the negative regulatory effector molecule that binds to this motif.

A CDw78 mAb FN1 was shown to recognize DP and/or DR molecules under the conditions of Western blotting. DP molecules were specifically retarded on a column of the FN1 immunosorbent; binding of FITC-labeled FN1 to B cell lines was completely blocked by excess of mAb to DR/DP beta chains, partially by several mAb to DP and weakly by some mAb to DR. The binding of two other CDw78 mAb, FN4 and MR11, to the B cell surface was most strongly inhibited by excess of different mAb to DR. Kinetics of stable binding of the CDw78 mAb indicated that their monovalent binding is of low affinity and that the stable binding to the surface is due to bivalent binding to two spatially close MHC class II molecules. FN1-based immunosorbent effectively immunoisolated complexes of MHC class II proteins with several tetraspanin molecules from a mild detergent lysate of a B cell line. It is concluded that FN1 and most likely also the other two CDw78 mAb recognize with low affinity determinants on MHC class II molecules (DP or DR) and preferentially bind in a stable fashion to dimerized or aggregated MHC class II molecules. Such dimers or aggregates may either exist as preformed on the cell surface or may be gradually formed and stabilized by bivalent interaction with mAb. These structures may be related to the previously described 'superdimers' of MHC class II and/or 'MHC-tetraspanin complexes'. CDw78 mAb may be valuable tools targeting such aggregated fraction of MHC class II molecules which can exhibit important signaling and antigen-presenting properties.

Four of the tetraspans family antigens expressed in B cells, CD37, CD53, TAPA-1, and R2/C33, as well as at least two other molecules, CD19 and CD21, coprecipitate with DR antigens from mild detergent lysates of human B-cell lines and tonsillar B cells. Coprecipitation and preclearing experiments indicate the existence of large multicomponent complexes containing jointly the seven components, although some "incomplete" complexes lacking some of the components may also exist. The complexes contain only a relatively small fraction of the total cellular pool of relevant molecules. The existence of these "tetraspans-DR complexes" may be related to the previously reported antiproliferative and signaling effects of antibodies against most of their components.

BALB/c, (BALB/c x B10.A)F1 and (BALB/c x B10)F1 hybrid mice were immunized with C-peptide of human proinsulin. The (BALB/c x B10.A)F1 hybrids were the best responders and yielded 3 hybridomas secreting specific monoclonal antibodies. One of them, C-PEP-01, bound the C-peptide with high affinity (Kas = 1.1 x 10(9) l/mol), cross-reacted fully with human proinsulin but not with insulin, glucagon or somatostatin and apparently recognized the regions of C-peptide comprising amino acid residues 8-13 and 25-31. A RIA system could be set up employing this monoclonal antibody suitable for estimation of C-peptide concentrations in a diagnostically useful range (1-50 ng/ml).

The previously demonstrated role of H-2 gene products in nonimmune cell interactions was further analyzed. As model of such interactions served the erythropoietic performance (in terms of 59Fe uptake) of bone marrow cell (BMC) inocula in the cellular environment of the spleen of massively irradiated hosts. When the performance in syngeneic hosts is taken as standard, the performance of cells from the same pool in H-2 disparate (congenic) hosts is subnormal. The inhibition was also observed in two congenic strain combinations where the H-2 disparity did not involve the I region. The capacity of BMC to distinguish H-2 -identical and H-2 -disparate hosts could be greatly reduced or virtually abolished by enzymatic digestion of certain cell surface carbohydrates. An essential effect of neuraminidase was further increased by emulsin. When the digested cells were left for 6 h in an enzyme-free medium, their capacity to dinstinguish H-2 disparities recovered. A pretreatment with papain, which cleaves a large fragment of the H-2 protein carrying both the antigenic site and the carbohydrate chain, had an effect comparable with but no greater than the sugar-removing enzymes. The enzymatic effect was largely due to the impairment of the performance of BMC in H-2 -identical hosts rather than to an improvement in H-2 -disparate ones. The neuraminidase- and emulsin-sensitive groupings thus seem to be essential for the cells to recognize “self” rather than “non-self” and to interact with H-2 -identical cells in physiological processes. A hypothesis is proposed that in H-2 glycoproteins, the antigenically and physiologically active sites are distinct, being located in the protein and carbohydrate parts of the molecules, respectively. It is further suggested that the normal function of the H-2 products might consist in providing the cell surface with structures which are complementary to similar structures on other cells; the carbohydrate moiety seems to be essential for this type of physiological interaction in contrast to the key and lock-type of complementarity in immune interactions which is based on protein structures.

Seven murine hybridomas were established producing IgG1 monoclonal antibodies (mAbs) against three different epitopes of human alpha-fetoprotein (alpha FP). The interaction of two of these mAbs (AFP-01 and AFP-02) with the antigen was strongly inhibited by calcium-chelating agents. The mAb AFP-11 could be used for estimation of alpha FP by RIA in the concentration range of 5-100 ng/ml. Several combinations of the mAbs could be used in a two-site sandwich-ELISA for similar purposes. Immunoaffinity chromatography on a column of immobilized mAb AFP-01 permitted the purification of alpha FP.

The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD 14, was determined by carboxypeptidase Y digestion and compared with the complete amino acid sequence of this protein as predicted from the structure of cloned cDNA [Goyert et al. Science239, 497–500 (1988)]. The soluble antigen isolated from urine appears to lack eight C-terminal amino acid residues predicted for the full-size translation product, but possesses a major part of the C-terminal hydrophobic domain originally suggested as the membrane-spanning segment. The CD 14 antigen can be removed from the monocyte surface by phosphatidylinositol-specific phospholipase C treatment, indicating that this glycoprotein is anchored in the membrane by a phospholipid and is not a transmembrane protein. The soluble form occurring in serum and in supernatants of cultured monocytes thus probably arises by phospholipase-mediated cleaving off the cell surface antigen. A sensitive sandwich ELISA was developed using a monoclonal anti-CD 14 antibody, MEM-18, and polyclonal rabbit anti-CD 14 antiserum for quantitation of the soluble antigen concns in sera and cell culture supernatants. Using this assay, the antigen present in the supernatant of phospholipase treated peripheral blood mononuclear cells could be estimated. The assay was also used for estimation of the concns of the soluble form of the CD 14 antigen in human sera.

Two monoclonal antibodies, MEM-15 and MEM-18, were prepared which recognize a monocyte 53-kDa antigen. A soluble form of this antigen was found in most normal sera and in urine of some nephrotic patients. Milligram amounts of this glycoprotein were isolated by immunoaffinity chromatography from urine and used for quantitative amino acid and carbohydrate analysis and N-terminal amino acid sequencing. The reactivity of the isolated antigen with several previously described monoclonal antibodies indicates that it is the antigen previously called MY-4 or MY-23. A significant homology exists between the sequenced N-terminal portion of the antigen and sequences found in the kinase-related transforming protein src and in the precursor of epidermal growth factor.

Nitrocellulose membranes were used as carriers of antigen for rapid screening of specific monoclonal antibodies or as carriers of the monoclonal antibody (in a crude form) for a simple and economic determination of isotype of the antibody by a modification of enzymoimmunoassay.

A convenient simple polyacrylamide gel electrophoresis system is described, suitable for semiquantitative analysis of the content of monoclonal antibodies in ascitic fluids of hybridoma-bearing mice.

The interaction of lectins with carbohydrate receptors on the plasma membrane of eukaryotic cells results in a wide variety of biological effects1. One effect which has been extensively studied is the stimulation of lymphocytes to blastogenesis and proliferation. High doses of concanavalin A (Con A) and phytohaemagglutinin (PHA) have been shown to activate in vitro regulatory cells capable of suppressing proliferation of other cells in the culture2,3 . However, Con A and PHA treatment were used effectively to prolong the survival of skin and heart allografts4–8 before it was recognised that some lectins have an activatory effect on suppressor cells in vitro. A possible explanation of these tolerogenic effects is the activation of specific suppressor cells. In this letter we have compared systematically various lectins differing in their carbohydrate specificity and mitogenicity in relation to their ability to induce prolonged skin allograft survival in mice with the aim of selecting the most effective lectin treatment schedule. Some preliminary results of this study have been mentioned in a recent review9.