Role of N-linked glycans of envelope glycoproteins in infectivity of human immunodeficiency virus type 1.

We have shown that enzymatic removal of N-linked glycans from human immunodeficiency virus type 1 (HIV-1) recombinant envelope glycoproteins gp160 and gp120 produced in BHK-21 cells did not significantly reduce their ability to bind to CD4, the cellular receptor for the virus. Because recombinant proteins may behave differently from proteins present on virions, we investigated whether such viral envelope glycoproteins either in a purified form or present on viral particles could be deglycosylated by treatment with an endoglycosidase F-N-glycanase mixture which cleaves all accessible glycan moieties. Endoglycosidase analysis of the carbohydrate composition of purified viral gp120 (vgp120) indicated a glycosylation pattern similar to that for recombinant gp120 (rgp120), and treatment with endoglycosidase F-N-glycanase resulted in comparable molecular weight (MW) reduction for both molecules. Similarly, after immunoblotting of the deglycosylated viral preparation, the characteristic 160- and 120-kilodalton (kDa) bands were replaced by 90- and 60-kDa bands, respectively. The apparent MW of gp41 shifted to 35 kDa. These results are consistent with complete deglycosylation. The immunoreactive conformation of envelope glycoproteins remained unaltered after deglycosylation: they were recognized to the same extent by specific human polyclonal or mouse monoclonal antibodies, and no proteolysis of viral proteins occurred during enzymatic treatment. Deglycosylation of vgp120 resulted in a less than 10-fold reduction of the ability to bind to CD4, presented either in a soluble form or at the cell membrane. In addition, deglycosylation significantly reduced, but did not abolish, HIV-1 binding to and infectivity of CD4+ cells as determined, respectively, by an indirect immunofluorescence assay and a quantitative dose-response infection assay. Taken together, these results indicate that removal of glycans present on mature envelope glycoproteins of HIV-1 diminishes but does not abolish either virus binding to CD4 or its capacity to infect CD4+ cells.     

Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor.

Tumor autocrine motility factor (AMF) has been detected in and purified from serum-free conditioned medium of human HT-1080 fibrosarcoma cells. Under nonreducing conditions, AMF migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of 55 kDa but under reducing conditions as a band of 64 kDa. Two-dimensional polyacrylamide gel electrophoresis of the purified AMF resolved two groups of polypeptides with isoelectric points of 6.1 and 6.2 (majors), 6.35 and 6.4 (minors). Purified AMF stimulated HT-1080 cell migration in a dose-dependent fashion. The motility stimulation of the fibrosarcoma cells with AMF is associated with the phosphorylation of the AMF receptor, a 78-kDa cell surface glycoprotein (gp78), suggesting protein kinase participation in migratory signal transduction. The gene encoding gp78 was cloned from an HT-1080 fibrosarcoma complementary DNA library. The deduced sequence encodes a polypeptide of 323 amino acids. The nucleotide and predicted amino acid sequence of the gp78 reveals significant homology with the human suppressor/oncogene p53 protein.     

gp160, the envelope glycoprotein of human immunodeficiency virus type 1, is a dimer of 125-kilodalton subunits stabilized through interactions between their gp41 domains.

The molecular masses, carbohydrate contents, oligomeric status, and overall molecular structure of the env glycoproteins of human immunodeficiency virus type 1--gp120, gp160, and gp41--have been determined by quantitative electron microscopy. Using purified gp160s, a water-soluble form of env purified from a recombinant vaccinia virus expression system, we have measured the masses of several hundred individual molecules by dark-field scanning transmission electron microscopy. When combined with sequence-based information, these mass measurements establish that gp160s is a dimer of subunits with an average monomer mass of 123 kDa, of which approximately 32 kDa is carbohydrate and 91 kDa is protein. Similarly, gp120 was found to be a monomer of 89 kDa and to contain virtually all of env's glycosylation. gp41 is glycosylated only slightly, if at all, and is responsible for the interactions that stabilize the gp160s dimer. A molecular mass map of gp160s derived by image processing depicts an asymmetric dumbbell whose two domains have masses of approximately 173 and approximately 73 kDa, corresponding to a gp120 dimer and a gp41 dimer, respectively. We infer that the average monomer mass of native gp160 is 125 kDa and that in situ, env is either a dimer or a tetramer but is most unlikely to be a trimer.     

Cell-mediated immune response toward viral envelope and core antigens in gibbon apes (Hylobates lar) chronically infected with human immunodeficiency virus-1

The specific cellular immune response toward envelope and core proteins of human immunodeficiency virus-1 (HIV-1) was investigated in gibbon apes chronically infected with the HTLV-IIIB isolate. After in vitro stimulation of PBMC from infected and control animals with HIV-1 Ag, DNA synthesis, IL-2R expression and IL-2 release were assayed. Cells from infected gibbon apes demonstrated a group-specific response toward whole virus preparations from three divergent HIV-1 isolates (HTLV-IIIB, HTLV-IIIRF, HTLV-IIIMN). Consistent responses were also detected against purified HIV-1 Ag, i.e., native gp120 envelope glycoprotein, recombinant gp160 glycoprotein, a synthetic peptide (peptide 7) representing a highly conserved region of gp120, and purified native core protein p24. In addition, lymphocytes from infected gibbon apes displayed a specific, MHC-restricted, cytotoxic activity against autologous cells expressing HIV-1 envelope or gag proteins. The specific T cell reactivity toward HIV-1 proteins observed in infected gibbons contrasts with findings in HIV-1 infected humans, and may help to explain the apparent discrepancy in the natural history of the infection between the two species.     

Human immunodeficiency virus type 1 envelope gp120 is cleaved after incubation with recombinant soluble CD4.

Human immunodeficiency virus type 1 (HIV-1) infects human CD4+ cells by a high-affinity interaction between its envelope glycoprotein gp120 and the CD4 molecule on the cell surface. Subsequent virus entry into the cells involves other steps, one of which could be cleavage of the gp120 followed by virus-cell fusion. The envelope gp120 is highly variable among different HIV-1 isolates, but conserved amino acid sequence motifs that contain potential proteolytic cleavage sites can be found. Following incubation with a soluble form of CD4, we demonstrate that gp120 of highly purified HIV-1 preparations is, without addition of exogenous proteinase, cleaved most likely in the V3 loop, yielding two proteins of 50 and 70 kDa. The extent of gp120 proteolysis is HIV-1 strain dependent and correlates with the recombinant soluble CD4 sensitivity to neutralization of the particular strain. The origin of the proteolytic activity in the virus preparations remains unclear. The results support the hypothesis that cleavage of gp120 is required for HIV infection of cells.     

Purification of a tumor-specific PNA-binding glycoprotein, gp200, from a human embryonal carcinoma cell line.

A 200-kDa peanut agglutinin (PNA)-binding glycoprotein, gp200, has been purified and partially characterized from the human embryonal carcinoma cell line, HT-E (833k). Tissue distribution analysis of this molecule by lectin blotting with PNA of detergent-extracted proteins from human cell lines and tissues demonstrated expression limited to nonseminomatous germ cell tumors. The 200-kDa protein was purified with lectin affinity and gel filtration chromatography. Purification to apparent homogeneity was demonstrated by one- and two-dimensional gel electrophoresis. Characterization of gp200 revealed it to be a surface integral membrane glycoprotein; however, gp200 could also be purified from the culture media of EC cells, suggesting gp200 has an extracellular role. The carbohydrate groups of gp200 are N-linked and partially sialylated and contain terminal galactose residues. These initial studies suggest that the PNA-defined glycoprotein, gp200, is a candidate for a nonseminomatous germ cell tumor marker.     

Galactose receptors and presentation of HIV envelope glycoprotein to specific human T cells.

Recognition of viral Ag and of the envelope glycoprotein of HIV (gp120) in particular by human Th cells is critical in the immune response to the viral Ag which includes antibody production and generation of cytotoxic cells. Procedures to increase antigenicity of gp120 are highly desirable in a vaccine perspective. Therefore, to induce activation of gp120-specific T cells by a liminal dose of Ag we enhanced uptake of gp120 by exploiting the galactose receptors on APC. Terminal sialic acid residues were removed by neuraminidase treatment from the carbohydrate side chains of the heavily glycosylated gp120. Galactose residues were exposed and hence recognized by galactose receptors on APC. The experiments demonstrated that 1) human monocytes and dendritic cells, but not cells of the B lineage, bear galactose receptor; 2) galactose receptors are indeed involved because enhanced presentation is inhibited by galactose and acetylgalactosamine and competed for by other asialoglycoproteins; 3) galactose receptors mediate internalization of Ag in intracellular compartments that intersect the processing and presenting pathways, resulting in activation of specific T cells; 4) antigenicity of gp120 for specific T cells can be enhanced by the exposure of galactose residues.     

Trypsin-sensitive and -resistant components in human T-cell membranes required for syncytium formation by human T-cell lymphotropic virus type 1-bearing cells.

Human T-cell lymphotropic virus type 1 (HTLV-1) envelope proteins play an important role in viral entry into target cells. In a syncytium formation assay consisting of a coculture of HTLV-1-bearing cells and target cells, mature gp46 and gp21 proteins each inhibited syncytium formation induced by HTLV-1-bearing cells. Experiments with 125I-labeled proteins showed that 125I-gp46 bound specifically with MOLT-4 target cells even in the presence of large amounts of gp21, whereas 125I-gp21 binding to target cells was completely blocked in the presence of large amounts of gp46. These observations suggest that HTLV-1 envelope proteins in syncytium formation interact with at least two components, which are located close to each other on the cell membrane. We isolated two components from MOLT-4 cell lysate, using Sepharose 4B columns coupled with peptides corresponding to amino acids 197 to 216 and 400 to 429, respectively, of the envelope protein. One is a trypsin digestion-sensitive component of approximately 34 to 35 kDa, which interacts specifically with gp46. The other is a nonprotein component, which interacts with gp21. This component was destroyed by sodium periodate oxidation and was partitioned into the methanol-chloroform phase. These observations suggest that these two components play an important role in HTLV-1 entry into target cells via membrane fusion.     

Challenge of chimpanzees (Pan troglodytes) immunized with human immunodeficiency virus envelope glycoprotein gp120.

The human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, infects humans and chimpanzees. To determine the efficacy of immunization for preventing infection, chimpanzees were immunized with gp120 purified from human T-cell lymphotrophic virus type IIIB (HTLV-IIIB)-infected cell membranes and challenged with the homologous virus, HTLV-IIIB. A challenge stock of HTLV-IIIB was prepared by using unconcentrated HTLV-IIIB produced in H9 cells. The titer of the virus from this stock on human and chimpanzee peripheral blood mononuclear cells and in human lymphoid cell lines was determined; a cell culture infectivity of 10(4) was assigned. All chimpanzees inoculated intravenously with 40 cell culture infectious units or more became infected, as demonstrated by virus isolation and seroconversion. One of two chimpanzees inoculated with 4 cell culture infectious units became infected. Chimpanzees immunized with gp120 formulated in alum developed antibodies which precipitated gp120 and neutralized HTLV-IIIB. Peripheral blood mononuclear cells from gp120-vaccinated and HIV-infected animals showed a significantly greater response in proliferation assays with HIV proteins than did peripheral blood mononuclear cells from nonvaccinated and non-HIV-infected chimpanzees. Two of the gp120-alum-immunized chimpanzees were challenged with virus from the HTLV-IIIB stock. One animal received 400 cell culture infectious units, and one received 40 infectious units. Both animals became infected with HIV, indicating that the immune response elicited by immunization with gp120 formulated in alum was not effective in preventing infection with HIV-1.     

Purification of a cell growth factor from a human lung cancer cell line: Its relationship with ferritin

We have purified a cell growth factor from a human lung cancer cell line, T3M-30, which was established in a protein-free chemically defined medium. The factor, designated carcinoma-derived growth factor (CD-GF), stimulated proliferation of a variety of cells, including human leukemia cells, HL-60, and melanoma cells, SK-28. Half-maximum stimulation by the purified CD-GF was achieved at a concentration of 40 ng/ml. In the purified CD-GF, two major protein bands of 24 kDa and 22 kDa were identified on a SDS polyacrylamide gel. The partial amino acid sequences of the 24 kDa protein were determined from two peptide fragments obtained by V8 protease treatment. The partial sequences were identical to those of heavy chain of human ferritin. The activity of the purified CD-GF was coprecipitated completely with a monoclonal antibody to heavy chain of ferritin. Ferritin has been considered to inhibit cell growth. However, human heart ferritin was capable of stimulating the growth of HL-60 cells. These results suggest that CD-GF is related to feritin and ferritin is a growth factor of HL-60 leukemia cells. © 1994 Wiley-Liss, Inc.     

C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.

C33 Ag was originally identified by mAb inhibitory to syncytium formation induced by human T cell leukemia virus type 1. The Ag was shown to be a highly heterogeneous glycoprotein consisting of a 28-kDa protein and N-linked oligosaccharides ranging from 10 to 50 kDa. In the present study, cDNA clones were isolated from a human T cell cDNA expression library in Escherichia coli by using mAb C33. The identity of cDNA was verified by immunostaining and immunoprecipitation of transfected NIH3T3 cells with mAb. The cDNA contained an open reading frame of a 267-amino acid sequence which was a type III integral membrane protein of 29.6 kDa with four putative transmembrane domains and three putative N-glycosylation sites. The C33 gene was found to belong to a newly defined family of genes for membrane proteins, such as CD9, CD37, CD53, CD63, and TAPA-1, and was identical to R2, a cDNA recently isolated because of its strong up-regulation after T cell activation. Availability of mAb for C33 Ag enabled us to define its distribution in human leukocytes. C33 Ag was expressed in CD4+ T cells, CD19+ B cells, CD14+ monocytes, and CD16+ granulocytes. Its expression was low in CD8+ T cells and mostly negative in CD16+ NK cells. PHA stimulation enhanced the expression of C33 Ag in CD4+ T cells by about 5-fold and in CD8+ T cells by about 20-fold. PHA stimulation also induced the dramatic size changes in the N-linked sugars previously shown to accompany human T cell leukemia virus type 1-induced transformation of CD4+ T cells.     

Isolation and characterization of leukosialin, a major sialoglycoprotein on human leukocytes

A major sialoglycoprotein (previously called gp105) on the human erythroleukemic cell line K562 was purified, and specific antibodies were raised in a rabbit. A number of different hematopoietic cell lines belonging to erythroid, myeloid, T-lymphoid, and B-lymphoid cell lineages were found to possess glycoproteins that were immunoprecipitated by these antibodies. However, the apparent molecular weights differed between cell lines, ranging from 113,000 to 150,000. In almost all cases, the immune precipitated molecule corresponded to the major sialoglycoprotein of the respective cell. Pulse-chase experiments showed that all cells produced an early precursor form of the molecule of 54 kDa, which was susceptible to endo-beta-N-acetylglucosaminidase H to give an apoprotein of 52 kDa. Neuraminidase treatment of the mature forms resulted in a characteristic decrease of the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (apparent molecular weights from 150,000 to 183,000). Amino acid analysis of the glycoprotein isolated from HL-60 cells showed a high content of serine, threonine, and proline, and the carbohydrate composition was compatible with the presence of a large number (approximately 90) of O-linked carbohydrate chains. The name leukosialin is proposed for this sialoglycoprotein, which seems to be widely distributed, but differently glycosylated, on leukocytes with diverse functions. In the following paper (Carlsson, S.R., Sasaki, H., and Fukuda, M. (1986) J. Biol. Chem. 261, 12787-12795), we demonstrate that the structures of O-linked oligosaccharides vary significantly depending on the cells from which leukosialin was isolated.     

Identification of a putative receptor for subgroup A feline leukemia virus on feline T cells.

Retrovirus infection is initiated by the binding of virus envelope glycoprotein to a receptor molecule present on cell membranes. To characterize a receptor for feline leukemia virus (FeLV), we extensively purified the viral envelope glycoprotein, gp70, from culture supernatants of FeLV-61E (subgroup A)-infected cells by immunoaffinity chromatography. Binding of purified 125I-labeled gp70 to the feline T-cell line 3201 was specific and saturable, and Scatchard analysis revealed a single class of receptor binding sites with an average number of 1.6 x 10(5) receptors per cell and an apparent affinity constant (Ka) of 1.15 x 10(9) M-1. Cross-linking experiments identified a putative gp70-receptor complex of 135 to 140 kDa. Similarly, coprecipitation of 125I-labeled cell surface proteins with purified gp70 and a neutralizing but noninterfering anti-gp70 monoclonal antibody revealed a single cell surface protein of approximately 70 kDa. These results indicate that FeLV-A binds to feline T cells via a 70-kDa cell surface protein, its presumptive receptor.     

Presence of oxidized protein hydrolase in human cell lines, rat tissues, and human/rat plasma

Oxidized protein hydrolase (OPH), an 80 kDa serine protease whose activity is inhibited by diisopropyl fluorophosphate (DFP), has been isolated from human erythrocytes [Fujino, T. et al. (1998) J. Biochem. 124, 1077-1085]. The presence of OPH in various biological samples was examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting using an anti-OPH antibody raised against OPH purified from human erythrocytes, and by [(3)H]DFP-labeling and successive SDS-PAGE/fluorography. Solubilized samples of human cell lines including K-562 cells, THP-1 cells and Jurkat cells, and rat tissues including brain, heart, liver, kidney, and testis, inhibited the anti-OPH antibody binding to OPH in ELISA. Immunoblotting of lysates of K-562 cells, THP-1 cells and Jurkat cells showed four immunoreactive protein bands including an 80 kDa protein. Immunoprecipitation of the [(3)H]DFP-labeled K-562 cell lysate and successive SDS-PAGE/fluorography showed the presence of only the 80 kDa DFP-reactive protein with OPH antigenic activity. The level of the 80 kDa immunoreactive protein in K-562 cells rose as the cells differentiated toward erythrocytes. Immunoblotting of human and rat plasma showed two immunoreactive protein bands, including the 80 kDa protein, and SDS-PAGE/fluorography of [(3)H]DFP-labeled rat and human plasma showed the presence of only the 80 kDa DFP-reactive protein. The results indicate that OPH is present in a wide variety of biological samples.     

The human T cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor: expression of a soluble form of gp39 with B cell co-stimulatory activity.

Signals delivered to B cells via CD40 can synergize with those provided by other B cell surface receptors to induce B cell proliferation and antibody class switching as well as modulate cytokine production and cell adhesion. Recently, it has been shown that the ligand for CD40 is a cell surface protein of approximately 39 kDa expressed by activated T cells, gp39. Here we report on the isolation and characterization of a cDNA clone encoding human gp39, a type II membrane protein with homology to TNF, and the construction and characterization of a soluble recombinant form of gp39. COS cell transfectants expressing gp39 synergized with either anti-CD20 mAb or PMA to drive strong B cell proliferation and alone were able to drive B cells to proliferate weakly. In all cases the B cell proliferation induced by gp39-expressing COS cells was reduced to background levels by the addition of soluble CD40. Unlike gp39-expressing COS cells, recombinant soluble gp39 was not mitogenic alone and required co-stimulation to drive B cell proliferation. These results suggest that B cells require a second signal besides gp39-CD40 to drive proliferation and that soluble gp39 alone in a non-membrane bound form is able to provide co-stimulatory signals to B cells.     

Enhancement of human melanoma antigen expression by IFN-beta

Although many immunotherapeutic investigations have focused on improving the effector limb of the antitumor response, few studies have addressed preventing the loss of tumor-associated Ag (TAA) expression, associated with immune escape by tumors. We found that TAA loss from human melanomas usually results from reversible gene down-regulation, rather than gene deletion or mutation. Previously, we showed that inhibitors of MAPK-signaling pathways up-regulate TAA expression in melanoma cell lines. We have now identified IFN-beta as an additional stimulus to TAA expression, including Melan-A/MART-1, gp100, and MAGE-A1. IFN-beta (but neither IFN-alpha nor IFN-gamma) augmented both protein and mRNA expression of melanocytic TAA in 15 melanoma lines (irrespective of initial Ag-expression levels). Treatment of low Ag melanoma lines with IFN-beta increased expression of melanocyte-lineage Ags, inducing susceptibility to lysis by specific CTLs. Treatment with IFN-beta also enhances expression of class I HLA molecules, thereby inducing both nominal TAA and the presenting HLA molecule. Data from fluorescent cellular reporter systems demonstrated that IFN-beta triggers promoter activation, resulting in augmentation of Ag expression. In addition to enhancing TAA expression in melanomas, IFN-beta also stimulated expression of the melanocytic Ag gp100 in cells of other neural crest-derived tumor lines (gliomas) and certain unrelated tumors. Because IFN-beta is already approved for human clinical use in other contexts, it may prove useful as a cotreatment for augmenting tumor Ag expression during immunotherapy.     

DF3 antigen, a human epithelial cell mucin, inhibits adhesion of eosinophils to antibody-coated targets

Although mucins provide lubrication and physical protection for epithelial cell surfaces, other functional roles for these large glycoproteins are unknown. One human mucin, designated DF3 Ag, is detectable on apical surfaces of normal epithelial secretory cells and in normal milk, urine, and plasma. The present studies have examined the effects of DF3 Ag purified from both normal and malignant sources on the antibody-dependent cytotoxicity of Schistosoma mansoni by eosinophils (ADCC-E) and on the adherence of eosinophils to inert antibody-coated targets. DF3 Ag purified from tissue culture supernatant of a human breast carcinoma cell line or from human milk inhibited ADCC-E in a concentration-dependent manner, with half-maximal inhibitory activity at 3 to 10 micrograms/ml. Inhibition of ADCC-E was specific for the DF3 mucin, because no inhibition was observed with two other unrelated, circulating glycoproteins: carcinoembryonic Ag and alpha 1-acid glycoprotein. Inhibition was not a result of direct cytotoxicity of the DF3 Ag for eosinophils, as demonstrated by the lack of detectable effect of the mucin on cellular trypan blue exclusion or PMA-induced H2O2 release. The inhibitory effect was time dependent, requiring the presence of DF3 Ag in the ADCC-E culture for at least 4 h, beginning within the first 2 h of eosinophil-schistosomula interaction. Furthermore, inhibition was not a result of interaction between DF3 Ag and the activating lymphokine. These data suggest that inhibition of ADCC-E by DF3 Ag is a result of interference of adhesion of eosinophils to Ig-coated targets. In this regard, purified DF3 tumor Ag prevents eosinophil adherence to human Ig-conjugated Sepharose 4B beads. Preincubation of the inert Ig-coated targets with DF3 Ag did not inhibit subsequent eosinophil adherence, suggesting that DF3 Ag interacts with a moiety present on the eosinophil. Inhibition of adhesion occurred at 37 degrees C, but was also observed at 4 degrees C. These results suggest that DF3 Ag acts as an immunomodulating agent. Because activated eosinophils can damage surrounding normal tissues as well as infectious organisms, DF3 Ag may serve to protect secretory epithelium from the cytotoxic effects of activated inflammatory cells.     

Immunochemical characterization of gp115, a yeast glycoprotein modulated by the cell cycle

A cell cycle-modulated glycoprotein (gp115, 115 kDa, isoelectric point 4.8-5) of Saccharomyces cerevisiae has been purified by Concanavalin A-affinity chromatography, followed by preparative two-dimensional gel electrophoresis, from yeast membrane proteins solubilized in Triton X-100. Antisera have been generated against the electrophoretically purified protein. Their specificity has been established by immunoblot analysis and by comparison of the partial proteolytic map obtained for the immunoprecipitated 35S-labeled 115 kDa polypeptide with that of the in vivo [35S]methionine-labeled gp115 isolated from two-dimensional gels. In tunicamycin-treated cells the immunoblot analysis identifies an unglycosylated precursor (86-88 kDa) and in sec18 mutant cells at the restrictive temperature an intermediary precursor of about 100 kDa. Six to seven carbohydrate chains have been estimated to be present on the gp115 protein, accounting for an electrophoretic shift corresponding to about 27 to 29 kDa of its relative molecular mass. Affinity-purified antibodies against the unglycosylated precursor (86-88 kDa) of gp115 were prepared and used to localize gp115 by indirect immunofluorescence microscopy. The similarity between the pattern of fluorescence obtained with these antibodies and that obtained using anti-plasma membrane H+-ATPase antibodies suggests an association of gp115 with the plasma membrane.     

Purification,localization, and expression of human intestinal alkaline sphingomyelinase

Sphingomyelin (SM) metabolism in the gut may have an impact on colon cancer development. In this study, we purified alkaline sphingomyelinase (alk-SMase) from human intestinal content, and studied its location in the mucosa, expression in colon cancer, and function on colon cancer cells. The enzyme was purified by a series of chromatographies. The molecular mass of the enzyme is 60 kDa, optimal pH is 8.5, and isoelectric point is 6.6. Under optimal conditions, 1 mg of the enzyme hydrolyzed 11 mM SM per hour. The properties of the enzyme are similar to those of rat intestinal alk-SMase but not to those of bacterial neutral SMase. Immunogold electronmicroscopy identified the enzyme on the microvillar membrane in endosome-like structures and in the Golgi complexes of human enterocytes. The expression and the activity of the enzyme were decreased in parallel in human colon cancer tissues compared with the adjacent normal tissue. The enzyme inhibited DNA biosynthesis and cell proliferation dose dependently and caused a reduction of SM in HT29 cells. Intestinal alk-SMase is localized in the enterocytes, down-regulated in human colon cancer, and may have antiproliferative effects on colon cancer cells.     

Retrovirally transduced human dendritic cells can generate T cells recognizing multiple MHC class I and class II epitopes from the melanoma antigen glycoprotein 100

Involvement of tumor-Ag specific CD4(+) and CD8(+) T cells could be critical in the generation of an effective immunotherapy for cancer. In an attempt to optimize the T cell response against defined tumor Ags, we previously developed a method allowing transgene expression in human dendritic cells (DCs) using retroviral vectors. One advantage of using gene-modified DCs is the potential ability to generate CD8(+) T cells against multiple class I-restricted epitopes within the Ag, thereby eliciting a broad antitumor immune response. To test this, we generated tumor-reactive CD8(+) T cells with DCs transduced with the melanoma Ag gp100, for which a number of HLA-A2-restricted epitopes have been described. Using gp100-transduced DCs, we were indeed able to raise T cells recognizing three distinct HLA-A2 epitopes within the Ag, gp100(154-162), gp100(209-217), and gp100(280-288). We next tested the ability of transduced DCs to raise class II-restricted CD4(+) T cells. Interestingly, stimulation with gp100-transduced DCs resulted in the generation of CD4(+) T cells specific for a novel HLA-DRbeta1*0701-restricted epitope of gp100. The minimal determinant of this epitope was defined as gp100(174-190) (TGRAMLGTHTMEVTVYH). These observations suggest that retrovirally transduced DCs have the capacity to present multiple MHC class I- and class II-restricted peptides derived from a tumor Ag, thereby eliciting a robust immune response against that Ag.