Increased immunogenicity of tumor vaccines complexed with anti-Gal: studies in knockout mice for alpha1,3galactosyltransferase. (25/1005)

A major prerequisite for the success of tumor vaccines is their effective uptake by antigen-presenting cells (APCs) and transport of these APCs to the draining lymph nodes, where the processed and presented tumor-associated antigens activate tumor-specific naive T cells. We previously suggested that the immunogenicity of autologus tumor vaccines in humans may be augmented by engineering vaccinating tumor cell membranes to express alpha-galactosyl (alpha-gal) epitopes (i.e., Galalpha1,3Galbeta1,4GlcNAc-R). Subsequent in situ binding of natural anti-Gal IgG molecules to these epitopes would result in the formation of immune complexes that target tumor vaccines for uptake by APCs, via the interaction of the Fc portion of anti-Gal with Fcgamma receptors on APCs. This hypothesis was tested in a unique experimental animal model of knockout mice for alpha1,3galactosyltransferase (alpha1,3GT) and the mouse melanoma B16-BL6 (referred to here as BL6). Like humans, these mice lack alpha-gal epitopes and produce anti-GaL BL6 melanoma cels are highly tumorigenic, and like human tumor cells, they lack alpha-gal epitopes. Expression of alpha-gal epitopes on these melanoma cells was achieved by stable transfection with alpha,3GT cDNA. The transfected melanoma cells (termed BL6alphaGT) express approximately 2 x 10(6) alpha-gal epitopes per cell and readily form immune complexes with anti-Gal. Vaccination of the mice with 2 x 10(6) irradiated melanoma cells that express alpha-gal epitopes, followed by challenge with 0.5 x 10(6) live parental melanoma cells, resulted in protection for at least 2 months (i.e, no tumor growth) in one-third of the mice, whereas all mice immunized with irradiated parental melanoma cells developed tumors 21-26 days post-challenge. The proportion of protected mice doubled when the mice were immunized twice with irradiated melanoma cells expressing alpha-gal epitopes and challenged with 0.2 x 10(6) live BL6 cells. Histological studies on the developing tumors in challenged mice that were immunized with melanoma cells expressing alpha-gal epitopes demonstrated extensive infiltration of T lymphocytes and macrophages, whereas no mononuclear cell infiltrates were observed in tumors of mice immunized with parental tumor cells. Overall, these studies imply that immunization of alpha1,3GT knockout mice with BL6 melanoma cells that express alpha-gal epitopes elicits, in a proportion of the population, protective immune response against the same tumor lacking such epitopes. These studies further suggest that similar immunization of cancer patients with autologous tumor vaccines that are engineered to express alpha-gal epitopes may increase the immune response to autologous tumor-associated antigens and, thus, may elicit immune-mediated destruction of metastatic cells expressing these antigens.  (+info)

Mixed chimerism induced without lethal conditioning prevents T cell- and anti-Gal alpha 1,3Gal-mediated graft rejection. (26/1005)

Gal alpha 1,3Gal-reactive (Gal-reactive) antibodies are a major impediment to pig-to-human xenotransplantation. We investigated the potential to induce tolerance of anti-Gal-producing cells and prevent rejection of vascularized grafts in the combination of alpha 1,3-galactosyltransferase wild-type (GalT(+/+)) and deficient (GalT(-/-)) mice. Allogeneic (H-2 mismatched) GalT(+/+) bone marrow transplantation (BMT) to GalT(-/-) mice conditioned with a nonmyeloablative regimen, consisting of depleting CD4 and CD8 mAb's and 3 Gy whole-body irradiation and 7 Gy thymic irradiation, led to lasting multilineage H-2(bxd) GalT(+/+) + H-2(d) GalT(-/-) mixed chimerism. Induction of mixed chimerism was associated with a rapid reduction of serum anti-Gal naturally occurring antibody levels. Anti-Gal-producing cells were undetectable by 2 weeks after BMT, suggesting that anti-Gal-producing cells preexisting at the time of BMT are rapidly tolerized. Even after immunization with Gal-bearing xenogeneic cells, mixed chimeras were devoid of anti-Gal-producing cells and permanently accepted donor-type GalT(+/+) heart grafts (>150 days), whereas non-BMT control animals rejected these hearts within 1-7 days. B cells bearing receptors for Gal were completely absent from the spleens of mixed chimeras, suggesting that clonal deletion and/or receptor editing may maintain B-cell tolerance to Gal. These findings demonstrate the principle that induction of mixed hematopoietic chimerism with a potentially relevant nonmyeloablative regimen can simultaneously lead to tolerance among both T cells and Gal-reactive B cells, thus preventing vascularized xenograft rejection.  (+info)

Human homolog of Caenorhabditis elegans sqv-3 gene is galactosyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. (27/1005)

A cDNA encoding a novel galactosyltransferase was identified based on BLAST analysis of expressed sequence tags, and the cDNA clones were isolated from a human melanoma line library. The new cDNA sequence encoded a type II membrane protein with 327 amino acid sequence and showed 38% homology to the Caenorhabditis elegans sqv-3 gene involved in the vulval invagination and oocyte development. Extracts from L cells transfected with the galactosyltransferase cDNA in an expression vector and a fusion protein with protein A exhibited marked galactosyltransferase activity specific for p-nitrophenyl-beta-D-xylopyranoside. Moreover, transfection with the cloned cDNA restored glycosaminoglycan synthesis of galactosyltransferase I-deficient Chinese hamster ovary mutant pgsB-761 cells. Analysis of the enzyme product by beta-galactosidase digestion, mass spectroscopy, and NMR spectroscopy revealed that the reaction product was formed via beta-1,4 linkage, indicating that the enzyme is galactosyltransferase I (UDP-galactose:O-beta-D-xylosylprotein 4-beta-D-galactosyltransferase, EC 2.4.1.133) involved in the synthesis of the glycosaminoglycan-protein linkage region of proteoglycans.  (+info)

Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus. (28/1005)

Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.  (+info)

Cloning and characterization of a close homologue of human UDP-N-acetyl-alpha-D-galactosamine:Polypeptide N-acetylgalactosaminyltransferase-T3, designated GalNAc-T6. Evidence for genetic but not functional redundancy. (29/1005)

The UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T3, exhibits unique functions. Specific acceptor substrates are used by GalNAc-T3 and not by other GalNAc-transferases. The expression pattern of GalNAc-T3 is restricted, and loss of expression is a characteristic feature of poorly differentiated pancreatic tumors. In the present study, a sixth human UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated GalNAc-T6, with high similarity to GalNAc-T3, was characterized. GalNAc-T6 exhibited high sequence similarity to GalNAc-T3 throughout the coding region, in contrast to the limited similarity that exists between homologous glycosyltransferase genes, which is usually restricted to the putative catalytic domain. The genomic organizations of GALNT3 and GALNT6 are identical with the coding regions placed in 10 exons, but the genes are localized differently at 2q31 and 12q13, respectively. Acceptor substrate specificities of GalNAc-T3 and -T6 were similar and different from other GalNAc-transferases. Northern analysis revealed distinct expression patterns, which were confirmed by immunocytology using monoclonal antibodies. In contrast to GalNAc-T3, GalNAc-T6 was expressed in WI38 fibroblast cells, indicating that GalNAc-T6 represents a candidate for synthesis of oncofetal fibronectin. The results demonstrate the existence of genetic redundancy of a polypeptide GalNAc-transferase that does not provide full functional redundancy.  (+info)

Cloning and expression of a proteoglycan UDP-galactose:beta-xylose beta1,4-galactosyltransferase I. A seventh member of the human beta4-galactosyltransferase gene family. (30/1005)

A seventh member of the human beta4-galactosyltransferase family, beta4Gal-T7, was identified by BLAST analysis of expressed sequence tags. The coding region of beta4Gal-T7 depicts a type II transmembrane protein with sequence similarity to beta4-galactosyltransferases, but the sequence was distinct in known motifs and did not contain the cysteine residues conserved in the other six members of the beta4Gal-T family. The genomic organization of beta4Gal-T7 was different from previous beta4Gal-Ts. Expression of beta4Gal-T7 in insect cells showed that the gene product had beta1,4-galactosyltransferase activity with beta-xylosides, and the linkage formed was Galbeta1-4Xyl. Thus, beta4Gal-T7 represents galactosyltransferase I enzyme (xylosylprotein beta1, 4-galactosyltransferase; EC 2.4.1.133), which attaches the first galactose in the proteoglycan linkage region GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. Sequence analysis of beta4Gal-T7 from a fibroblast cell line of a patient with a progeroid syndrome and signs of the Ehlers-Danlos syndrome, previously shown to exhibit reduced galactosyltransferase I activity (Quentin, E., Gladen, A., Roden, L., and Kresse, H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1342-1346), revealed two inherited allelic variants, beta4Gal-T7(186D) and beta4Gal-T7(206P), each with a single missense substitution in the putative catalytic domain of the enzyme. beta4Gal-T7(186D) exhibited a 4-fold elevated K(m) for the donor substrate, whereas essentially no activity was demonstrated with beta4Gal-T7(206P). Molecular cloning of beta4Gal-T7 should facilitate general studies of its pathogenic role in progeroid syndromes and connective tissue disorders with affected proteoglycan biosynthesis.  (+info)

Negative regulation of oligodendrocyte differentiation by galactosphingolipids. (31/1005)

Galactocerebroside and sulfatide, major galactosphingolipid components of oligodendrocyte plasma membranes and myelin, are first expressed at a critical point, when progenitors cease to proliferate and commence terminal differentiation. We showed previously that an antibody to galactocerebroside/sulfatide arrested terminal differentiation, suggesting a role for these galactolipids in oligodendrocyte differentiation. We have now investigated the differentiation of oligodendrocytes (1) in response to other anti-galactolipid antibodies, showing that anti-sulfatide O4 but not anti-galactocerebroside O1 blocks terminal differentiation, perhaps by mimicking an endogenous ligand, and (2) in a transgenic mouse unable to synthesize these lipids because of mutation of the gene for ceramide galactosyltransferase, a key enzyme for galactosphingolipid synthesis. We find that galactosyltransferase mRNA expression begins at the late progenitor [pro-oligodendroblast (Pro-OL)] stage of the lineage and that the late progenitor marker pro-oligodendroblast antigen is not synthesized in the absence of galactosyltransferase. The principal outcome of the elimination of these galactolipids is a two- to threefold enhancement in the number of terminally differentiated oligodendrocytes both in culture and in vivo. Because the general pattern of differentiation and the level of progenitor proliferation and survival appear to be unaltered in the mutant cultures, we conclude that the increased number of oligodendrocytes is caused by an increased rate and probability of differentiation. In agreement with these two experimental approaches, we present a model in which galactosphingolipids (in particular galactocerebroside and/or sulfatide) act as sensors and/or transmitters of environmental information, interacting with endogenous ligands to function as negative regulators of oligodendrocyte differentiation, monitoring the timely progress of Pro-OLs into terminally differentiating, myelin-producing oligodendrocytes.  (+info)

Molecular basis for the progeroid variant of Ehlers-Danlos syndrome. Identification and characterization of two mutations in galactosyltransferase I gene. (32/1005)

Progeroid type Ehlers-Danlos (E-D) syndrome was reported to be caused by defects in galactosyltransferase I (EC 2.4.1.133), which is involved in the synthesis of common linkage regions of proteoglycans. Recently, we isolated cDNA of the galactosyltransferase I (XGalT-1) (Okajima, T., Yoshida, K., Kondo, T., and Furukawa, K. (1999) J. Biol. Chem. 274, 22915-22918). Therefore, we analyzed mutations in this gene of a patient with progeroid type E-D syndrome by reverse transcription polymerase chain reaction and direct sequencing. Two changes of G and T to A and C at 186 and 206, respectively, were detected. Then, we determined the genomic DNA sequences encompassing the A186D and L206P mutations, revealing that the unaffected parents and two siblings were heterozygous for either one of the two different mutations and normal, while the patient had both of two different mutant genes. Enzymatic functions of cDNA clones of XGalT-1 containing the individual mutations were examined, elucidating that L206P clone completely lost the activity, while A186D retained approximately 50% or 10% of the activity when analyzed with extracts from cDNA transfectant cells or recombinant soluble enzymes, respectively. Moreover, L206P enzyme showed diffuse staining in the cytoplasm of transfectant cells, while the wild type or A186D clones showed Golgi pattern. These results indicated that the mutations in XGalT-1 were at least one of main molecular basis for progeroid type E-D syndrome.  (+info)