Enzyme assays, using extract or bovine 1,3galactosyltransferase as the enzyme source, were done in a 25 l reaction combination containing 0
Enzyme assays, using extract or bovine 1,3galactosyltransferase as the enzyme source, were done in a 25 l reaction combination containing 0.5 mM UDP-[14C]-Gal (6 Ci/mol) (Amersham Biosciences, Buckinghamshire, UK), 20 mM MnCl2, 4 mM ATP, 0.5% Triton X-100, 100 mM Na-cacodylate buffer, pH 7.2, and 1 mM acceptor substrate. induce protective immunity by vaccination (Vervelde et al., 2002; Knox et al., 2003; Redmond and Knox, 2006). Several native BMS-191095 antigens, including hidden gut-derived antigens, can induce protection against (Knox et al., 2003). However, attempts to induce protection employing recombinant forms of these antigens are not encouraging, suggesting that specific post-translational modifications, such as glycosylation, may contribute to the protective properties of these proteins (Vervelde et al., 2002). Glycosylation can greatly contribute to the immunogenicity of proteins, especially when the glycans are foreign to the host. Glycans are abundant on the surface and secretory products of helminths, and are well exposed to the environment. Both glycans of BMS-191095 the parasitic trematode (Okano et al., 1999, 2001) and nematode-glycans (Tawill et al., 2004) have the capacity to trigger T-helper 2 (Th2) type responses and the production of glycan-specific antibodies in their hosts (Okano et al., 1999, 2001). Individuals infected with species and chimpanzees immunized with radiation-attenuated cercariae showed high levels of anti-glycan serum IgG to the glycan antigens GalNAc1-4(Fuc1-2Fuc1-3)GlcNAc (LDN-DF) and Fuc1-3GalNAc1-4GlcNAc (F-LDN), glycan motifs that are not found in mammals (van Remoortere et al., 2001, 2003a, 2003b; van Die and Cummings, 2006). Recent data showed that vaccination with natural excretory/secretory (ES) antigens from in Alhydrogel, a strong Th2 type response-inducing adjuvant, induced protection in lambs against challenge infection with ES antigens induces multiple anti-glycan antibodies, the same sera as used in our previous studies were screened on a glycan-array containing more than 250 different glycan antigens. The data show that vaccination of lambs with ES antigens indeed resulted in eliciting multiple anti-glycan antibodies, which varied depending on the adjuvant used. In addition to anti-LDNF IgG, a high level of IgG realizing the glycan antigen Gal1-3GalNAc was observed only in sera of the guarded lambs, which were vaccinated with ES antigens in Alhydrogel. Our data revealed that glycoproteins from different developmental stages of contain a terminal Gal1-3GalNAc-R moiety, a glycan antigen that to our knowledge has not been reported before on helminth glycoproteins. 2. Materials and methods 2.1. Materials Sera from lambs were obtained from studies explained previously (Vervelde et al., 2003). Essentially, Black Bless sheep were immunized s.c. three times at 3 week BMS-191095 intervals (at day 0, day 21 and day 42) with L3s. ES Rabbit polyclonal to EGFR.EGFR is a receptor tyrosine kinase.Receptor for epidermal growth factor (EGF) and related growth factors including TGF-alpha, amphiregulin, betacellulin, heparin-binding EGF-like growth factor, GP30 and vaccinia virus growth factor. antigens were obtained as previously explained (Vervelde et al., 2003). The lectin GSI-B4-biotin was purchased from Sigma (St. Louis, MO, USA). Goat anti-mouse-peroxidase (PO), streptavidin-PO and streptavidin-alkalic phosphatase were purchased from Jackson Immunoresearch (West Grove, USA). The anti-mouse-alkalic phosphatase was purchased from Zymed laboratories, Inc. (San Francisco, USA) and mouse anti-sheep IgG was from Serotec (Kidlington, UK). The anti-Gal1-3Gal antibody M86 (Galili et al., 1998) was a kind gift from Dr. U. Galili (University or college of Massachusetts Medical School, USA). Monocytes were isolated from buffycoat (Sanquin, Amsterdam, the Netherlands) with CD14 MACS beads (Miltenyi biotec, Auburn, USA) according to the manufacturers protocol. Gal1-3Gal-polyacrylamide (PAA), Gal1-3GalNAc-PAA and glucitol-PAA were purchased from Lectinity (~20% substitution, Lectinity, Finland) and LDNF-BSA was synthesized as previously explained (van Remoortere et al., 2000). p-Nitrophenyl-N-acetyl–D-GalNAc (GalNAc-pNP), GalNAc-pNP, Gal-pNP, Gal-pNP, Gal1-4GlcNAc-pNP (LN-pNP) were purchased from Sigma (St. Louis, MO, USA). GalNAc1-4GlcNAc-O-(CH2)8COOCH3 was a kind gift from Ole Hindsgaul (University or college of Alberta, Canada). Fuc1-2Gal1-3GlcNAc-O(CH2)7CH3, Fuc1-2Gal1-4GlcNAc-O-(CH2)8COOCH3 and Gal1-3GlcNAc-O-(CH2)8COOCH3 were a kind gift from Monica Palcic (University or college of Alberta, Canada). 2.2. Glycan array Glycan array screening was performed by Core H of the Consortium for Functional Glycomics (CFG) (University or college of Oklahoma, Oklahoma, USA). The glycan array is usually a microarray made up of a library of natural and synthetic glycans with amino linkers printed onto (adults and L3s), (adults and L3s), (L3s and ES antigens), (adults), (adults), (adults), (adults and cercariae) as explained by De Bose-Boyd et al. (1998). For Western blotting, frozen worms were thawed and resuspended in 100 BMS-191095 mM Tris-HCl, pH 8, made up of protease inhibitors. For ELISA assays, the proteins of the helminth homogenates were precipitated by adding 4 vol. of (-20C) acetone. Subsequently, the combination was incubated for 1 h at -20C, the protein pellet collected by centrifugation for 10 min at 13,000 and re-suspended in.