Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A natural antibody with binding specificity for recombinant tissue-type plasminogen activator (t-PA) expressed in mouse C127 cells was present in almost all disease-free humans and patients with thrombotic disease examined. This antibody was specific for a carbohydrate, alpha 1-3-linked galactose residue, and was isolated by affinity chromatography using Synsorb 90 coupled with the glycosidic epitope Gal alpha 1-3Gal beta 1-4Glc-R as an immunoadsorbent. The evaluation of various glycoproteins for ability to bind the purified antibody in ELISA demonstrated that not only recombinant t-PA from C127 cells but also recombinant erythropoietin (EPO) and recombinant protein C produced in C127 cells have alpha 1-3-linked galactose residues on their sugar side chains. This anti-alpha-galactosyl antibody also interacted with natural t-PA from human vascular trees (vascular t-PA) and placenta (placenta t-PA), but not to melanoma t-PA, recombinant t-PA, EPO or protein C expressed in Chinese hamster ovary (CHO) cells.
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PMID:Specificity of human natural antibody to recombinant tissue-type plasminogen activator (t-PA) expressed in mouse C127 cells. 211 46

Carbohydrates are T cell independent antigens because they do not bind to MHC molecules. However, glycopeptides might potentially bind to MHC molecules via their peptide component for presentation to T cells. We have conjugated the disaccharide galabiose [Gal alpha (1-4)Gal beta] to the amino terminus of a T cell peptide determinant from hen egg-white lysozyme [HEL(52-61)]. The resulting glycopeptide (Gal2-52-61) and a nonglycosylated analogue containing tyrosine and glutamic acid at the amino-terminus (YE-52-61) bound equally well to purified I-Ak. T cell hybridomas were produced after immunization with Gal2-52-61. Many of the T cell hybridomas were glycopeptide-specific and responded to Gal2-52-61 but not to nonglycosylated synthetic peptides or to HEL presented by APC, indicating that the carbohydrate moiety influenced T cell recognition. Recognition was lost with the amino terminal attachment of the disaccharide to a peptide six amino acids longer at the amino terminus than HEL(52-61). Recognition also was lost with peptides containing only a single galactosyl residue or with galabiose bound to a different I-Ak binding peptide. T cells directed to Gal2-52-61 recognized glycopeptides having significant variation in the disaccharide structure, such as HEL(52-61) glycopeptides carrying lactose, cellobiose, or hepta-o-acetylated galabiose. Peptide residues were important features of the T cell epitope; Ala substitutions of two critical T cell contact residues of HEL(52-61) (Tyr53 and Leu56) abrogated T cell reactivity to the glycopeptides without affecting binding to I-Ak. In conclusion, we propose that these T cells recognize a peptide conformation specific to glycopeptide-I-Ak complexes and that this recognition does not involve specific interaction between the carbohydrate moiety and the T cell receptor.
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PMID:Glycopeptides bind MHC molecules and elicit specific T cell responses. 836 Apr 71

The alpha-gal epitope (Galalpha1-3Galbeta1-(3)4GlcNAc-R) is abundantly synthesized on glycolipids and glycoproteins of non-primate mammals and New World monkeys by the glycosylation enzyme alpha1,3galactosyltransferase (alpha1,3GT). In humans, apes and Old World monkeys, this epitope is absent because the alpha1,3GT gene was inactivated in ancestral Old World primates. Instead, humans, apes and Old World monkeys produce the anti-Gal antibody, which specifically interacts with alpha-gal epitopes and which constitutes approximately 1% of circulating immunoglobulins. Anti-Gal has functioned as an immunological barrier, preventing the transplantation of pig organs into humans, because anti-Gal binds to the alpha-gal epitopes expressed on pig cells. The recent generation of alpha1,3GT knockout pigs that lack alpha-gal epitopes has resulted in the elimination of this immunological barrier. Anti-Gal can be exploited for clinical use in cancer immunotherapy by targeting autologous tumour vaccines to APC, thereby increasing their immunogenicity. Autologous intact tumour cells from haematological malignancies, or autologous tumour cell membranes from solid tumours are processed to express alpha-gal epitopes by incubation with neuraminidase, recombinant alpha1,3GT and with uridine diphosphate galactose. Subsequent immunization with such autologous tumour vaccines results in in vivo opsonization by anti-Gal IgG binding to these alpha-gal epitopes. The interaction of the Fc portion of the vaccine-bound anti-Gal with Fcgamma receptors of APC induces effective uptake of the vaccinating tumour cell membranes by the APC, followed by effective transport of the vaccinating tumour membranes to the regional lymph nodes, and processing and presentation of the tumour-associated antigen (TAA) peptides. Activation of tumour-specific T cells within the lymph nodes by autologous TAA peptides may elicit an immune response that in some patients will be potent enough to eradicate the residual tumour cells that remain after completion of standard therapy. A similar expression of alpha-gal epitopes can be achieved by transduction of tumour cells with an adenovirus vector (or other vectors) containing the alpha1,3GT gene, thus enabling anti-Gal-mediated targeting of the vaccinating transduced cells to APC. Intratumoral delivery of the alpha1,3GT gene by various vectors results in the expression of alpha-gal epitopes. Such expression of the xenograft carbohydrate phenotype is likely to induce anti-Gal-mediated destruction of the tumour lesion, similar to rejection of xenografts by this antibody. Opsonization of the destroyed tumour cell membranes by anti-Gal IgG further targets them to APC, thus converting the tumour lesion, treated by the alpha1,3GT gene, into an in situ autologous tumour vaccine.
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PMID:The alpha-gal epitope and the anti-Gal antibody in xenotransplantation and in cancer immunotherapy. 1626 20

Dendritic cells (DC) are professional APC that control the balance between T cell immunity and tolerance. Genetic engineering of DC to regulate the outcome of the immune response is an area of intense research. Galectin (gal)-1 is an endogenous lectin that binds to glycoproteins and exerts potent regulatory effects on T cells. Consequently, gal-1 participates in central deletion of thymocytes and exerts therapeutic effects on experimental models of T cell-mediated autoimmune disorders and graft-vs-host disease. Together, these observations strongly indicate that engineering DC to express transgenic (tg) gal-1 may be beneficial to treat T cell-mediated disorders. In this study, we have investigated the impact of the expression of high levels of tg gal-1 on maturation/activation of DC and on their T cell stimulatory function. Murine DC were transduced with a recombinant adenovirus encoding hu gal-1 (gal-1-DC). Tg gal-1 was exported by a nonclassical pathway through exosomes and was retained on the DC surface inducing segregation of its ligand CD43. Expression of tg gal-1 triggered activation of DC determined by induction of a more mature phenotype, increased levels of mRNA for proinflammatory cytokines, and enhanced ability to stimulate naive T cells. Conversely, gal-1-DC induced rapid apoptosis of activated T cells. In vivo, gal-1-DC increased significantly the sensitization phase of contact hypersensitivity assays while inducing a drastic inhibition of the elicitation phase by triggering apoptosis of activated T cells in the dermis. Gal-1-DC represent a novel tool to control differentially the afferent and efferent arms of the T cell response.
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PMID:Transgenic galectin-1 induces maturation of dendritic cells that elicit contrasting responses in naive and activated T cells. 1675 64

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.
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PMID:Intratumoral injection of alpha-gal glycolipids induces xenograft-like destruction and conversion of lesions into endogenous vaccines. 1737 27

BJ-48, a serine protease from the venom of Bothrops jararacussu, was purified to homogeneity using affinity chromatography on p-aminobenzamidine-agarose followed by HPLC gel filtration. BJ-48 presented 52kDa by SDS-PAGE analysis and 48,036Da by electron spray mass spectrometry. The enzyme was shown to be highly glycosylated with 42% of N-linked carbohydrates composed of Fuc(1):GalN(4):GlcN(5):Gal(1):Man(2) and a high content of sialic acid residues (8-12%). BJ-48 had optimal esterase activity at pH 7.5 and displayed maximum catalytic rate at 50 degrees C. Its hydrolytic activity was strongly inhibited by aprotinin and dithiothreitol while N-tosyl-l-phenylalanine chloromethyl ketone, 6-aminocaproic acid, E-64 and soybean trypsin inhibitor (SBTI) were ineffective. The kinetics of BJ-48 with chromogenic substrates revealed an unprecedented selectivity (10(4)-fold) for Arg over Lys in P1. BJ-48 proved to be a thrombin-like enzyme (TLE) with a specific fibrinogen-clotting activity of 73.4NIH units/mg. The TLE rapidly digested human fibrinogen Bbeta chain, but the Aalpha chain was cleaved specifically to release fibrinopeptide A with k(cat)/K(m)=2.1 microM(-1)s(-1). The TLE showed no activity toward other thrombin substrates like protein C, protease-activated receptor-1 or inhibitors such as hirudin and antithrombin. A non-denaturing procedure using PNGase F and neuraminidase followed by hydrophobic interaction chromatography was employed to obtain active BJ-48 forms with variable carbohydrate content. Compared to the native enzyme, total or partially deglycosylated BJ-48 forms presented up to 2-fold reduction in their specific activities upon heating at 55/65 degrees C or treatment with SBTI. These results point out a role for BJ-48 glycosylation in thermostability and controlling the access of some canonical protein inhibitors to the active site.
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PMID:BJ-48, a novel thrombin-like enzyme from the Bothrops jararacussu venom with high selectivity for Arg over Lys in P1: Role of N-glycosylation in thermostability and active site accessibility. 1743 97

Constitutive activation of the Wnt pathway as a result of APC, AXIN1 or CTNNB1 mutations has been found in most colorectal cancers. For a long time, this aberrant Wnt activation has been thought to be independent of upstream signals. However, recent studies indicate that upstream signals retain their ability to regulate the Wnt pathway even in the presence of downstream mutations. Wnt-2 is well known for its overexpression in colorectal cancer. Galectin-3 (Gal-3), a multifunctional carbohydrate binding protein implicated in a variety of biological functions, has recently been reported to interact with beta-catenin. In this study, we investigated roles of Wnt-2 and Gal-3 in the regulation of canonical Wnt/beta-catenin signaling. We found that siRNA silencing of either Wnt-2 or Gal-3 expression inhibited TCF-reporter activity, decreased cytosolic beta-catenin level and induced apoptosis in human colorectal cancer cells containing downstream mutations. More interestingly, we showed that inhibition of both Wnt-2 and Gal-3 had synergistic effects on suppressing canonical Wnt signaling and inducing apoptosis, suggesting that aberrant canonical Wnt/beta-catenin signaling in colorectal cancer can be regulated at multiple levels. The combined inhibition of Wnt-2 and Gal-3 may be of superior therapeutic advantage to inhibition by either one of them, giving rise to a potential development of novel drugs for the targeted treatment of colorectal cancer.
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PMID:Inhibition of Wnt-2 and galectin-3 synergistically destabilizes beta-catenin and induces apoptosis in human colorectal cancer cells. 1753 95

alpha-Gal glycolipids capable of converting tumors into endogenous vaccines, have alpha-gal epitopes (Gal alpha 1-3 Gal beta 1-4GlcNAc-R) and are extracted from rabbit RBC membranes. alpha-Gal epitopes bind anti-Gal, the most abundant natural antibody in humans constituting 1% of immunoglobulins. alpha-Gal glycolipids insert into tumor cell membranes, bind anti-Gal and activate complement. The complement cleavage peptides C5a and C3a recruit inflammatory cells and APC into the treated lesion. Anti-Gal further opsonizes the tumor cells and targets them for effective uptake by recruited APC, via Fc gamma receptors. These APC transport internalized tumor cells to draining lymph nodes, and present immunogenic tumor antigen peptides for activation of tumor specific T cells. The present study demonstrates the ability of alpha-gal glycolipids treatment to prevent development of metastases at distant sites and to protect against tumor challenge in the treated mice. Adoptive transfer studies indicate that this protective immune response is mediated by CD8+ T cells, activated by tumor lesions turned vaccine. This T cell activation is potent enough to overcome the suppressive activity of Treg cells present in tumor bearing mice, however it does not elicit an autoimmune response against antigens on normal cells. Insertion of alpha-gal glycolipids and subsequent binding of anti-Gal are further demonstrated with human melanoma cells, suggesting that intratumoral injection of alpha-gal glycolipids is likely to elicit a protective immune response against micrometastases also in cancer patients.
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PMID:Intratumoral injection of alpha-gal glycolipids induces a protective anti-tumor T cell response which overcomes Treg activity. 1918 2

Anti-Gal constitutes approximately 1% of circulating IgG in humans and interacts specifically with alpha-gal epitopes. We reported previously that expression of alpha-gal epitopes on HIV gp120 and influenza virus vaccines increases immunogenicity by approximately 100-fold. We hypothesize that immunogenicity of any microbial vaccine can be markedly increased by linked alpha-gal epitopes due to in vivo formation of immune complexes with anti-Gal and the effective internalization of such immune complexes by APC, via Fc/FcgammaR interaction. The increased transport to lymph nodes and processing of anti-Gal complexed vaccines internalized by APC, results in effective activation of vaccine specific CD4(+) and CD8(+) T cells, and high cellular and humoral immune response. This universal mechanism for anti-Gal mediated increased immunogenicity is demonstrated in alpha1,3galactosyltransferase knockout mice with ovalbumin as a model vaccine.
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PMID:Mechanism for increased immunogenicity of vaccines that form in vivo immune complexes with the natural anti-Gal antibody. 1942 21

Developing an effective HIV-1 vaccine will require strategies to enhance antigen presentation to the immune system. In a previous study we demonstrated a marked increase in immunogenicity of the highly glycosylated HIV-1 gp120 protein following enzymatic addition of alpha-gal epitopes to the carbohydrate chains. In the present study we determined whether gp120(alphagal) can also serve as an effective platform for targeting other HIV-1 proteins to APC and thus increase immunogenicity of both proteins. For this purpose we produced a recombinant fusion protein between gp120 and the HIV-1 matrix p24 protein (gp120/p24). Multiple alpha-gal epitopes were synthesized enzymatically on the gp120 portion of the fusion protein to generate a gp120(alphagal)/p24 vaccine. Immune responses to gp120(alphagal)/p24 compared to gp120/p24 vaccine lacking alpha-gal epitopes were evaluated in alpha1,3galactosyltransferase knockout (KO) mice. These mice lack alpha-gal epitopes and, therefore, are capable of producing the anti-Gal antibody. T cell responses to p24, as assessed by ELISPOT and by CD8+ T cells intracellular staining assays for IFNgamma, was on average 12- and 10-fold higher, respectively, in gp120(alphagal)/p24 immunized mice than in mice immunized with gp120/p24. In addition, cellular and humoral immune responses against gp120 were higher by 10-30-fold in mice immunized with gp120(alphagal)/p24 than in gp120/p24 immunized mice. Our data suggest that the alpha-gal epitopes on the gp120 portion of the fusion protein can significantly augment the immunogenicity of gp120, as well as that of the fused viral protein which lacks alpha-gal epitopes. This strategy of anti-Gal mediated targeting to APC may be used for production of effective HIV-1 vaccines comprised of various viral proteins fused to gp120.
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PMID:Increased immunogenicity of HIV-1 p24 and gp120 following immunization with gp120/p24 fusion protein vaccine expressing alpha-gal epitopes. 2003 7


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