Mapping the Anopheles gambiae odorant binding protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays.

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.

Characterization of a membrane-associated glycoprotein (gp 43) on human hepatocellular carcinoma by a monoclonal antibody.

A monoclonal antibody, Hepama-1, produced by immunizing mice with cells of a human hepatocellular carcinoma cell line, has been used to identify and characterize a previously unreported antigen present on the surface of human hepatocellular carcinoma cells. The antigen occurred on the membranes of human hepatoma cell lines and tumor biopsies but was not detectable in tumors of other origin or normal tissues. Binding was determined by enzyme-linked immunoabsorbent assay and immunofluorescence on cell lines and by immunoperoxidase staining of tissue sections. In immunofluorescence studies, Hepama-1 antibodies stained five out of six human hepatoma cell lines, showed only slight binding to breast tumor cell lines, but failed to stain colon tumor or normal cell lines. The antihepatoma antibody exhibited positive immunoperoxidase staining of human liver tumor sections but did not stain tumors of other origin. Hepama-1 bound specifically to a membrane glycoprotein with an approximate molecular weight of 43,000. Western blot and solid phase enzyme-linked immunoabsorbent assay analysis showed that the 43-kD antigen occurred on five of six human hepatoma cell lines and was expressed by every human hepatocellular carcinoma biopsy tested. This cell surface molecule represents a potentially useful target for immunotherapy and localization of human hepatocellular carcinomas.

Enhanced expression of ganglioside GD3 in human and rat hepatocellular carcinoma cells and NIH 3T3 cells transfected with human tumor DNAs.

The gangliosides of human hepatoma biopsies, human hepatoma cell lines, and diethylnitrosamine-induced rat hepatomas were examined. These malignant tissues all expressed increased content of disialolactosylceramide (GD3) with respect to their normal counterparts. During the induction of rat hepatoma by diethylnitrosamine, an increase in GD3 levels appeared as early as 12 wk after initiation of diethylnitrosamine, concurrent with the appearance of precancerous hepatocytes. GD3 levels gradually increased to a peak of 4 times that of normal rat liver at 20 wk. CMP-NeuAc:GM3 sialyltransferase, the enzyme that synthesizes GD3 by transfer of sialic acid to GM3, also had tumor-associated elevation during the course of diethylnitrosamine-induction of rat hepatomas. To investigate the relationship of oncogene transformation and changes in ganglioside biosynthesis, NIH 3T3 cells transfected DNAs from human hepatoma or nasopharyngeal carcinoma were studied. The transfectants each expressed the same ganglioside composition, including a detectable level of GD3, as well as enhanced activity of CMP-NeuAc:GM3 sialyltransferase. A correlation between the tumor DNA transfection and the augmentation of GD3 in malignant cells is discussed. Because of the early appearance of GD3 in hepatoma and its possible relationship to oncogene activation, GD3 may be a potentially useful early tumor marker.

Rapid analysis of membrane glycopeptides by gel permeation high-performance liquid chromatography.

A rapid procedure for the analysis of glycopeptides has been developed using gel permeation high-performance liquid chromatography (HPLC). Glycopeptides derived by exhaustive pronase digestion of glycoproteins from radiolabeled human tumor and normal cell lines were chromatographed on DuPont GF-250 and GF-450 gel permeation columns in buffers containing non-ionic detergents. Effective separations of glycopeptides ranging in molecular mass from less than 600 daltons to more than 20,000 daltons, equivalent to the separation range of Sephadex G50 chromatography, were achieved in 7 min. The separations were dependent upon the use of an isocratic mobile phase, that contained a low-ionic-strength Tris buffer and Nonidet P-40 or Triton X-100. The mobilities of protein standards indicated the occurrence of a biphasic elution system, which favored the separation of species with molecular masses below 20,000 daltons. Glycopeptides isolated by this method could be applied directly to lectin or ion-exchange columns or could be digested with neuraminidase, endo H or other enzymes without further treatment. Removal of sialic acid from the glycopeptides caused a dramatic increase in retention time. Using this method, glycopeptides could be isolated rapidly and in high yield. The ease, speed and reproducibility of the separations and compatibility of the solvent systems with affinity or ion-exchange chromatography techniques make this gel permeation HPLC method an ideal initial step in the purification of glycopeptides.

Analysis of ras oncogene products by two-dimensional gel electrophoresis: evidence for protein families with distinctive molecular forms.

Protein products of the ras family of oncogenes were immunoprecipitated by an anti-p21 monoclonal antibody, separated by two-dimensional gel electrophoresis and subsequently detected by western immunoblot analysis using the same anti-p21 monoclonal antibody as a probe. Using this method, a 21 kDa oncogene protein (p21) was detected and characterized in cell lines containing Harvey (Ha), Kirsten (Ki), neuroblastoma (N), or cellular (proto) ras genes. The ras gene products from all cell types occurred with multiple forms differing in size, charge or in both parameters. Transforming ras oncogene proteins occurred in easily identifiable groups that were different from each other in molecular weight and charge, were distinctive for each ras gene type and were different from cellular ras equivalents. Similar, but not identical, family groups occurred in different cell types containing the same oncogene. The reproducible occurrence of unpredicted p21 forms suggests that previously unreported post-translational processing steps may be associated with the synthesis of certain oncogene products. This immunoprecipitation/two-dimensional gel/western blot technique is a simple method for the identification and characterization of p21 gene products.

Isolation and characterization of a monoclonal antibody specific for epithelial cells.

Monoclonal antibodies were generated to antigens on human foreskin keratinocytes to identify epithelial-specific molecules. Spleen cells from BALB/c mice, immunized with membrane preparations from primary explants of foreskin epithelial cells, were fused with the NS-1 mouse myeloma line. Hybridoma supernatants were screened for the desired immunological reactivity using enzyme-linked immunosorbant binding assays. Hybridomas secreting antibodies reacting with epithelial cells, but not fibroblasts or lymphocytes, were cloned by limiting dilution, and two stable clones producing immunoglobulin M K antibodies were selected for study. Evaluation of fixed paraffin-embedded human tissue by an indirect immunoperoxidase technique revealed that the antibodies bound most strongly to normal stratified squamous and transitional epithelium, and squamous and transitional cell carcinomas. Antibodies from the cloned hybridomas also reacted with primary cell cultures of foreskin keratinocytes, pulmonary epithelium, fetal liver, and amnion cells, but not with primary cultures of nonepithelial cells. Further testing by enzyme-linked immunosorbent assays revealed that the antibodies reacted with some long-term cell lines derived from epithelial tumors. Nonepithelial cell lines were not stained by the antibodies. Indirect immunofluorescent studies indicated that staining was confined to the cell surface. These antibodies may prove useful in studies of differentiation markers of human epithelial cells.

The anticarcinogenic and tumor growth inhibitory activities of lymphotoxin are associated with altered membrane glycoprotein synthesis.

Membrane glycoprotein synthesis was studied in non-tumorigenic and tumor cells treated with the lymphokine hormone, lymphotoxin, to define the biochemical mechanisms by which lymphotoxin prevents carcinogenesis and inhibits growth of tumor cells. Syrian hamster lymphotoxin increased the synthesis of high molecular weight (50,000-250,000) membrane glycoproteins in non-tumorigenic secondary passage NIH/N Syrian hamster embryo fibroblasts and caused a decrease in the synthesis of high molecular weight glycoproteins in benzo[a]pyrene-induced hamster tumor cells. Increased glycoprotein synthesis varied directly with lymphotoxin concentration and duration of treatment and occurred contemporaneously with lymphotoxin-initiated prevention of carcinogen-induced morphologic transformation. These lymphotoxin-stimulated fluctuations in membrane glycoprotein synthesis are evidence of specific biochemical target cell changes associated with the anticarcinogenic and tumor-inhibitory actions of this immunologic hormone.

Identification of beta-lymphotoxin as the predominant molecular class of in vitro and in vivo Syrian hamster lymphotoxin.

The molecular class of Golden Syrian hamster lymphotoxin produced in vitro and in vivo was determined by size-exclusion high-performance liquid chromatography using silica-based protein separation columns eluted with a 0.1 M sodium phosphate, pH 7.4 buffer containing 0.1% Mr 4000 polyethylene glycol. Lymphotoxin cytolytic activity was quantitated in the column effluent by measuring the ability of the fractions to lyse alpha-L929 cells as indicated by [3H]TdR release. Lymphotoxin activity induced by an 8- or 24-hr or 5-day phytohemagglutinin stimulation of peritoneal leukocytes, by 24-hr phytohemagglutinin-coated alpha-L929-cell stimulation of peritoneal leukocytes, or by 24-hr phytohemagglutinin stimulation of spleen cells occurred in the Mr range of 20,000-56,000, with major components in the 35,000-50,000 beta-lymphotoxin region. No activity was present in the complex (greater than 200,000) region and only minimal activity was detectable in the alpha (70,000-160,000) and gamma (12,000-20,000) regions. In vivo-induced lymphotoxin, obtained by peritoneal lavage 48 hr after intraperitoneal administration of phytohemagglutinin, was entirely beta-lymphotoxin and was not detectable in the plasma. Lymphotoxin produced in vitro and injected simultaneously with the gamma-emitting radionuclide 99mtechnetium, inhibited in vivo development of radiation-induced transplacental carcinogenesis. Thus, Syrian hamster lymphotoxin with antitumor activity consists of glycoproteins with isoelectric points of 4.8-5.2, Mr of 20,000-56,000, and major in vitro and in vivo forms in the beta-lymphotoxin range.

Rapid separation of biologically active Syrian hamster lymphotoxin in high yield by size-exclusion high-performance liquid chromatography.

A rapid, simple and reproducible high-performance liquid chromatography (HPLC) procedure is described for the partial purification of biologically active Syrian hamster lymphotoxin. Lymphotoxin, a series of lymphocyte derived immunological glycoprotein hormones with molecular weights of 20,000 to 56,000, has been partially purified by a single-step HPLC procedure or by a two-step isoelectric focusing-HPLC procedure. The HPLC separation method uses silica-based protein separation columns eluted with a 0.1 M sodium phosphate, pH 7.4, 0.1% polyethylene glycol 4000 buffer at a flow-rate of 0.5 ml/min at room temperature. Nearly complete recoveries of biologically active lymphotoxin can be obtained with as much as a 13-fold purification in less than 1 h.

Biochemical characterization of human melanoma cell surfaces: dissection with monoclonal antibodies.

This report describes the structures of three distinct human melanoma surface antigens detected by monoclonal anti-melanoma antibodies. One antigen, expressed on all melanoma cells and on certain astrocytoma cells but not on any other kind of normal or tumor cell tested, has been shown to consist of our associated polypeptide chains. A second antigen expressed on many but not all melanomas has been identified as the antigenic product of the HLA-D locus, the DR antigen. A third protein antigen is not found on any normal cell tested but occurs on some, but not all, tumors of various origins.

Glycopeptides derived from individual membrane glycoproteins from control and Rous sarcoma virus-transformed hamster fibroblasts.

The membrane glycoproteins from control (BHK21/C13) and Rous sarcoma virus-transformed (C13/B4) baby hamster kidney cells grown in medium containing [14C]- or D-[3H]glucosamine have been separated into two distinct classes: a phenol-soluble fraction and an aqueous fraction. The membrane glycoproteins from both BHK21/C13 and C13/B4 partitioned similarly into these two fractions. The phenol and aquesous-soluble glycoproteins differed in their sodium dodecyl sulfate-polyacrylamide gel profiles, polyacrylamide isoelectric focusing profiles, and glycopeptide distribution on Sephadex G-50. A number of aqueous and phenol-soluble glycoproteins from BHK21/C13 and C13/B4 cells were purified to near homogeneity by means of polyacrylamide electrophoresis and gel electrofocusing. These glycoproteins range in molecular weight from 179,000 to 31,000 and have isoelectric points of 7.5 to 3.0. Our results show that the pronase glycopeptides of 20 out of 24 homologous membrane glycoproteins of equivalent molecular weight and isoelectric point from BHK21/C13 and C13/B4 cells are dissimilar as measured by Sephadex G-50 gel filtration.

Surface glycoproteins of normal and transformed cells: a difference determined by sialic acid and a growth-dependent sialyl transferase.

The pattern of elution from a column of Sephadex G-50 of a fucose-labeled carbohydrate component derived from the surface glycoproteins of transformed cells can be altered to resemble that from untransformed cells by enzymatic removal of the sialic acid. These results indicate that the consistent differences found between control and virus-transformed cells may depend upon a relatively specific sialyl transferase that is found in greater amounts (2.5- to 11-times) in transformed cells than in control cells, and in dividing cells as compared to nondividing cells.