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• W2018172904 abstract "Core fucosylation of N-linked oligosaccharides (GlcNAcβ1,4(Fucα1,6)GlcNAcβ1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideumglycoproteins (Crandall, I. E. and Newell, P. C. (1989)Development 107, 87–94), specifically recognize fucose residues in α1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing α-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core α1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice. Core fucosylation of N-linked oligosaccharides (GlcNAcβ1,4(Fucα1,6)GlcNAcβ1-Asn) is a common modification in animal glycans, but little is known about the distribution of core-fucosylated glycoproteins in mammalian tissues. Two monoclonal antibodies, CAB2 and CAB4, previously raised against carbohydrate epitopes of Dictyostelium discoideumglycoproteins (Crandall, I. E. and Newell, P. C. (1989)Development 107, 87–94), specifically recognize fucose residues in α1,6-linkage to the asparagine-bound GlcNAc of N-linked oligosaccharides. These IgG3 antibodies do not cross-react with glycoproteins containing α-fucoses in other linkages commonly seen in N- or O-linked sugar chains. CAB4 recognizes core α1,6 fucose regardless of terminal sugars, branching pattern, sialic acid linkage, or polylactosamine substitution. This contrasts to lentil and pea lectins that recognize a similar epitope in only a subset of these structures. Additional GlcNAc residues found in the core of N-glycans from dominant Chinese hamster ovary cell mutants LEC14 and LEC18 progressively decrease binding. These antibodies show that many proteins in human tissues are core-fucosylated, but their expression is localized to skin keratinocytes, vascular and visceral smooth muscle cells, epithelia, and some extracellular matrix-like material surrounding subpopulations of lymphocytes. The availability of these antibodies now allows for an extended investigation of core fucose epitope expression in development and malignancy and in genetically manipulated mice. l-Fucosyl residues in α1,6-linkage to the innermost GlcNAc (“core fucose”) are relatively common in mammalian N-glycans. The enzyme GDP-l-fucose:2-acetamido-2-deoxy-β-d-glucoside (Fuc→Asn-linked GlcNAc) 6-α-l-fucosyltransferase, which catalyzes the transfer of l-fucose from GDP-fucose to the Asn-linked GlcNAc, has been purified from human skin fibroblasts (1Voynow J.A. Kaiser R.S. Scanlin T.F. Glick M.C. J. Biol. Chem. 1991; 266: 21572-21577Abstract Full Text PDF PubMed Google Scholar) and substrate specificity studied in the porcine liver enzyme (2Longmore G.D. Schachter H. Carbohydr. Res. 1982; 100: 365-392Crossref PubMed Scopus (156) Google Scholar). The enzyme has recently been cloned from porcine brain (3Uozumi N. Yanagidani S. Miyoshi E. Ihara Y. Sakuma T. Gao C.-X. Teshima T. Fujii S. Shiba T. Tanuguchi N. J. Biol. Chem. 1996; 271: 27810-27817Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). A great deal of attention has been drawn to the modifications near the nonreducing terminus of oligosaccharides with the success of demonstrating biological functions, e.g. sialyl Lewisx in selectin binding (4Bevilacqua M.P. Nelson R.M. J. Clin. Invest. 1993; 91: 379-387Crossref PubMed Scopus (1063) Google Scholar), mannose 6-phosphate in lysosomal enzyme targeting (5Kornfeld S. FASEB J. 1987; 1: 462-468Crossref PubMed Scopus (351) Google Scholar), sialic acids in protein recognition (6Varki A. FASEB J. 1997; 11: 248-255Crossref PubMed Scopus (491) Google Scholar), polysialic acids in neuronal development (7Tang J. Rutishauser U. Landmesser L. Neuron. 1994; 13: 405-414Abstract Full Text PDF PubMed Scopus (238) Google Scholar), and N-acetylgalactosamine 4-sulfate in pituitary hormonal regulation (8Baenziger J.U. Kumar S. Brodbeck R.M. Smith P.L. Beranek M.C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 334-338Crossref PubMed Scopus (160) Google Scholar). In contrast, the biological significance of core modifications in N-glycans has not been clearly elucidated. The presence of a core fucose residue greatly enhances recognition of N-linked sugar chains by lentil (Lens culinarisagglutinin) and pea (Pisum sativum agglutinin) lectins (9Kornfeld K. Reitman M.L. Kornfeld R. J. Biol. Chem. 1981; 256: 6633-6640Abstract Full Text PDF PubMed Google Scholar). Bourne et al. (10Bourne Y. Mazurier J. Legrand D. Rouge P. Montreuil J. Spik G. Cambillau C. Structure. 1994; 2: 209-219Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 11Bourne Y. Rouge P. Cambillau C. J. Biol. Chem. 1992; 267: 197-203Abstract Full Text PDF PubMed Google Scholar) demonstrated that core fucose binds within a small crevice of Lathyrus ochrus isolectin II, but in its absence the Manα1,3Man arm of the oligosaccharide is in an energetically less favorable conformation that prevents strong binding. Thus fucose promotes the glycan to assume the critical conformation required for lectin binding. More recently, Stubbs et al.(12Stubbs H.J. Lih J.J. Gustafson T.L. Rice K.G. Biochemistry. 1996; 35: 937-947Crossref PubMed Scopus (69) Google Scholar) showed that core fucose greatly influences the conformation and flexibility of the Manα1,6Man antenna of the biantennary oligosaccharide from porcine fibrinogen. These studies suggest that core fucose residues could play important roles in defining oligosaccharide conformations needed for specific carbohydrate-protein interactions. For example, core fucosylation is required for polysialylation of neural cell adhesion molecule by the specific polysialic acid synthase (13Kojima N. Tachida Y. Yoshida Y. Tsuji S. J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar) and is involved in regulation of de-N-glycosylation by mammalian peptide N-glycosidases (14Suzuki T. Seko A. Kitajima K. Inoue Y. Inoue S. J. Biol. Chem. 1994; 269: 17611-17618Abstract Full Text PDF PubMed Google Scholar). The expression of many oligosaccharides is known to be highly regulated in a tissue- and cell-specific manner, reflecting the differential regulation of glycosyltransferases (15Dinter A. Berger E.G. Adv. Exp. Med. Biol. 1995; 376: 53-82Crossref PubMed Scopus (18) Google Scholar). Enhanced core fucosylation of proteins such as α1-fetoprotein and α1-protease inhibitor in germ cell tumors, hepatocellular carcinomas, and other neoplasms (16Aoyagi Y. Isemura M. Yosizawa Z. Suzuki Y. Sekine C. Ono T. Ichida F. Biochim. Biophys. Acta. 1985; 830: 217-223Crossref PubMed Scopus (137) Google Scholar, 17Aoyagi Y. Suzuki Y. Isemura M. Nomoto M. Sekine C. Igarashi K. Ichida F. Cancer (Phila.). 1988; 61: 769-774Crossref PubMed Scopus (160) Google Scholar, 18Aoyagi Y. Suzuki Y. Igarashi K. Yokota T. Mori S. Suda T. Naitoh A. Isemura M. Asakura H. Cancer (Phila.). 1993; 72: 615-618Crossref PubMed Scopus (21) Google Scholar, 19Goodarzi M.T. Turner G.A. Clin. Chim. Acta. 1995; 236: 161-171Crossref PubMed Scopus (52) Google Scholar) suggests that this modification may be restricted in normal human tissues. However, little is known about the tissue distribution of core-fucosylated glycoproteins in humans. The literature is replete with histochemical studies that use lectins to detect glycoconjugate expression in tissues (for recent reviews see Refs. 20Danguy A. Akif F. Pajak B. Gabius H.-J. Histol. Histopathol. 1994; 9: 155-171PubMed Google Scholar, 21Spicer S.S. Schulte B.A. J. Histochem. Cytochem. 1992; 40: 1-38Crossref PubMed Scopus (386) Google Scholar, 22Walker R.A. Pathol. Res. Pract. 1989; 185: 826-835Crossref PubMed Scopus (51) Google Scholar). Cytochemical staining obtained with L. culinaris agglutinin and P. sativum agglutinin is considered as chiefly indicating the presence of core-fucosylated glycans, although fucosylation only serves to enhance binding of these lectins to the trimannosyl core of complex oligosaccharides. Most of the lectin histochemistry studies of adult and embryonic mammalian tissues include L. culinaris agglutinin and P. sativum agglutinin as part of lectin “mixtures” (23Bell C.M. Skerrow C.J. Br. J. Dermatol. 1984; 111: 517-526Crossref PubMed Scopus (44) Google Scholar, 24Nemanic M.K. Whitehead J.S. Elias P.M. J. Histochem. Cytochem. 1983; 31: 887-897Crossref PubMed Scopus (130) Google Scholar, 25Truong L.D. Phung V.T. Yoshikawa Y. Mattioli C.A. Histochemistry. 1988; 90: 51-60Crossref PubMed Scopus (54) Google Scholar, 26Capaldi M.J. Dunn M.J. Sewry C.A. Dubowitz V. Histochem. J. 1985; 17: 81-92Crossref PubMed Scopus (31) Google Scholar, 27Nakagawa F. Schulte B.A. Spicer S.S. Cell Tissue Res. 1986; 245: 579-589Crossref PubMed Scopus (32) Google Scholar, 28Zachariah B. Marikar Y. Basu D. Indian J. Biochem. Biophys. 1991; 28: 412-417PubMed Google Scholar), but in most cases the binding patterns have been similar to those obtained with concanavalin A. Lectin binding studies also have other inherent shortcomings, since many lectins with the same nominal specificity show different staining intensities for the same cell or tissue structure (29Damjanov I. Lab. Invest. 1987; 57: 5-20PubMed Google Scholar). Monoclonal antibodies are more sensitive and specific than lectins, but many of the established carbohydrate-specific monoclonal antibodies are low affinity IgM types with significant cross-reactivities. IgG monoclonal antibodies with increased specificity and sensitivity would be more advantageous for in situ localization of oligosaccharides in tissues and for detection by immunoassays. Also, with the advent of new in vivo genetic approaches for elucidating oligosaccharide function (30Varki A. Marth J. Semin. Dev. Biol. 1995; 6: 127-138Crossref Scopus (80) Google Scholar), transgenic expressions or deletions of glycosyltransferases require high quality reagents to assess tissue-specific distribution of oligosaccharides. IgG monoclonal antibodies that recognize specific linkages should have a decided advantage over lectins that are often defined by their monosaccharide specificities. During our study of a library of carbohydrate-specific monoclonal antibodies made against Dictyostelium discoideumglycoproteins, we found two IgG antibodies that specifically recognized fucose residues linked α1,6 to the Asn-bound GlcNAc of N-linked oligosaccharides. We used these antibodies to study the expression of core-fucosylated glycoconjugates in human tissues, and we find that they may have a much more restricted cell type localization than previously believed. Fucosylated BSA 1The abbreviations used are: BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; HRP, horseradish peroxidase; PLA2, phospholipase A2; pFg, porcine fibrinogen; pTg, porcine thyroglobulin; EPO, erythropoietin; CHO, Chinese hamster ovary; BCIP/NBT, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium. neoglycoproteins were generously provided by Dr. Ole Hindsgaul of the University of Alberta, Edmonton, Alberta, Canada. They were prepared and analyzed for sugar content as described previously (31Lemieux R.U. Baker D.A. Weinstein W.M. Switzer C.M. Biochemistry. 1981; 20: 199-205Crossref PubMed Scopus (54) Google Scholar, 32Kamath V.P. Diedrich P. Hindsgaul O. Glycoconj. J. 1996; 13: 315-319Crossref PubMed Scopus (118) Google Scholar).tert-Butoxycarbonyl-l-tyrosine oligosaccharides from porcine fibrinogen (pFg) and reducing oligosaccharides from recombinant erythropoietin (EPO) were generous gifts from Dr. Kevin Rice, University of Michigan. They were characterized by proton NMR and fast atom bombardment-mass spectrometry or a combination of two-dimensional high pressure liquid chromatography mapping and enzymatic digestions (33Da Silva M.L.C. Tamura T. McBroom T. Rice K.G. Arch. Biochem. Biophys. 1994; 312: 151-157Crossref PubMed Scopus (28) Google Scholar, 34Rice K. Takahashi N. Namiki Y. Tran A.D. Lisi P.J. Lee Y.C. Anal. Biochem. 1992; 206: 278-287Crossref PubMed Scopus (61) Google Scholar). Horseradish peroxidase (HRP), honeybee (Apis mellifera) venom phospholipase A2(PLA2), pineapple stem bromelain, ovalbumin, pFg, porcine thyroglobulin (pTg), human lactoferrin, human α1-acid glycoprotein, polyclonal rabbit anti-HRP, HRP-agarose, PLA2agarose, Aspergillus β-xylosidase, biotinylated L. culinaris agglutinin, P. sativum agglutinin, biotinylated anti-mouse IgG, avidin peroxidase, and immunoglobulin isotyping kit were purchased from Sigma. Biotinylated Ulex europeus agglutinin I lectin was from Vector Laboratories, Burlingam, CA. Streptavidin-biotin kit was from Dako, Carpenteria, CA. Goat anti-mouse IgG alkaline phosphatase conjugate was from Promega, Madison WI. L. culinaris agglutinin-alkaline phosphatase conjugate was obtained from E-Y Laboratories, San Mateo, CA. Chicken liver α-l-fucosidase was from Oxford Glycosystems, NY. Lumiphos 530 was from Lumigen Inc. Southfield, MI. Proteinase K was obtained from Boehringer Mannheim. Human tissues were obtained from the Tissue Core Facility of the Cancer Center, University of California, San Diego. Production of monoclonal antibodies CAB2 and CAB4 against cell surface proteins of D. discoideum was described earlier (35Crandall I.E. Newell P.C. Development. 1989; 107: 87-94PubMed Google Scholar). Immunoglobulin isotyping was done as per the manufacturer's instructions. Reference glycoproteins or fucosylated BSA conjugates were immobilized on 96-well microtiter plates, and the wells were blocked with 3% BSA in Tris-HCl saline (TBS) overnight. They were washed and the antigens then allowed to react with the CAB antibodies at concentrations of 4 μg/ml IgG, in TBS containing 1% BSA and 0.1% Tween 20 for 1 h at room temperature. The plates were then washed and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG, followed by development with p-nitrophenyl phosphate substrate. They were read at 405 nm on an ELISA plate reader. pFg was coated onto the wells of FluoroNunc Maxi-sorb plates and blocked with 1% gelatin in phosphate-buffered saline (PBS). The wells were incubated with CAB4 at a concentration of 250 ng/ml in TBS containing 1% BSA and 0.2% Tween 20 for 2 h at 37 °C, followed by incubation with alkaline phosphatase-conjugated goat anti-mouse IgG. The plates were then developed with Lumiphos-530 and were read on an Anthos-LUCY1 luminometer. Human tissues were homogenized with a BioHomogenizer in 50 mm Tris-HCl, pH 7.5, containing 0.1 m2-mercaptoethanol and 1% SDS. Suspensions were centrifuged at 650 × g for 15 min, and the postnuclear supernatants were harvested and centrifuged further for 30 min at 100,000 ×g. After protein estimation, the supernatants were stored frozen until analysis. Cell lysates from LEC10, LEC14, LEC18, and Lec13 CHO cell mutants were kindly provided by Dr. Pamela Stanley, Albert Einstein College of Medicine, New York, NY. Cell extracts were made in 1.5% Triton X-100, and postnuclear supernatants were analyzed by CAB4 in immunoblots. Proteins were separated by SDS-polyacrylamide gel electrophoresis in 12.5% polyacrylamide gels under reducing conditions and transferred to nitrocellulose membranes. The membranes were blocked overnight with 10% skimmed milk in TBS or 3% BSA in TBS, washed with TBS containing 0.05% Tween 20, and incubated with either of the CAB antibodies at concentrations of 4 μg/ml IgG for reference proteins, 1 μg/ml for human tissue extracts, and 400 ng/ml for CHO cell lysates or with 2.5 μg to 5 μg/ml L. culinarisagglutinin-alkaline phosphatase conjugate for 1 h at room temperature. For the immunoblots this was followed by reaction with alkaline phosphatase-conjugated goat anti-mouse IgG. Bound proteins were visualized by incubating with 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate. Antibodies to HRP are predominantly directed against core modifications on its N-glycans, specifically Xylβ1,2Manβ-, and Fucα1,3GlcNAcβ-Asn. Commercial rabbit polyclonal anti-HRP (purified IgG fraction) was affinity purified on a column of HRP-agarose and further fractionated into anti-Fucα1,3GlcNAc and anti-Xylβ1,2Manβ- components by a second affinity purification on a PLA2 column as described (36Faye L. Gomord V. Fitchette-Laine A.C. Chrispeels M.J. Anal. Biochem. 1993; 209: 104-108Crossref PubMed Scopus (153) Google Scholar). The bound anti-fucose component reacted with all plant glycoproteins carrying Fucα1,3GlcNAc in the core, and with PLA2 which also carries the same modification, but it did not recognize Fucα1,6GlcNAcβ in the core of mammalian N-glycans. The anti-xylose component that was isolated from the run-through fraction of the PLA2 column was repurified on the same column and was found to be completely free of the anti-fucose reactivity assayed with PLA2. Details of the purification and characterization of these antibodies will be described elsewhere. One μg of honey bee venom PLA2 or pFg were each incubated with 4 milliunits of chicken liver α-l-fucosidase at 37 °C for 20 h, in a total volume of 50 μl in 100 mm citrate/phosphate buffer, pH 6.0. A control tube containing each protein was incubated without added enzyme. (The digestion was done with and without prior denaturation of the proteins using 0.1% SDS, 100 °C for 2 min, and the results were essentially the same for both treatments.) After heat inactivation of the enzyme (100 °C, 5 min), the control and digested proteins were tested for binding to CAB4 antibody using ELISAs. In addition, control and digested PLA2 were also tested against the anti-Fucα1,3GlcNAc component of anti-HRP to determine the specificity of the enzyme. One μg of HRP was incubated with 50 milliunits of Aspergillus β-xylosidase at 37 °C for 16 h, in 50 μl of 100 mm phosphate buffer, pH 6.0, containing 100 μm dithiothreitol. A control was incubated without added enzyme. After heat inactivation of the enzyme, the control and digested proteins were tested for binding to CAB4 using an ELISA. Efficiency of digestion was monitored by checking loss of reactivity of the digested protein with the purified anti-Xylβ1,2Manβ- fraction of anti-HRP. Core α1,3-fucosylated glycopeptides, core α1,6-fucosylated glycopeptides, and non-fucosylated glycopeptides from HRP, pTg/pFg, and ovalbumin respectively, were prepared from 50 mg of each protein by digesting with 2.5–5 mg of proteinase K in 0.2 m Tris-HCl buffer, pH 7.5, for 24 h. The reaction mixture was boiled for 10 min and centrifuged. The glycopeptides were lyophilized and purified on a Bio-Gel P2 column equilibrated with 0.1 m ammonium formate, pH 6.0. Fractions were assayed for neutral sugar, and void fractions were pooled and repeatedly lyophilized from water to remove ammonium formate. The glycopeptides were then reconstituted in water. Neutral sugar was measured by phenol sulfuric acid method, and total sugar concentration was calculated from the established structure of N-linked oligosaccharides from each protein. Cryostat sections of human tissues (5-micron thickness) were cut and air-dried. Sections were fixed in 10% buffered formalin for 20 min followed by removal of the endogenous peroxidase with 0.03% hydrogen peroxide if necessary, and by blocking of nonspecific binding sites with 10% normal goat serum in PBS containing 1% BSA. Five-micron paraffin sections were deparaffinized and rehydrated before proceeding with the immunostaining. After washing, the antibodies were overlayered onto serial tissue sections at predetermined dilutions (usually between 1 and 10 μg/ml), and the slides were incubated in a humid atmosphere for 30 min at room temperature or overnight at 4 °C. The labeled streptavidin biotin kit from Dako was used as per the manufacturer's instructions or with PBS or TBS washes between every step, and biotinylated anti-mouse IgG was then applied for 10 min followed by either alkaline phosphatase or peroxidase-linked streptavidin for 10 min. After another wash, the appropriate substrate was added, and the slides were incubated in the dark for 20 min. After a wash in buffer, they were counterstained with hematoxylin, mounted, and viewed using an Olympus BH2 microscope. Lectin staining was carried out using biotinylated U. europeus agglutinin I, L. culinaris agglutinin, or P. sativum agglutinin. Incubation with the lectins was carried out in TBS containing 1% BSA and 1 mm CaCl2, MgCl2, and MnCl2, followed by alkaline phosphatase-conjugated streptavidin and Fast Red as the developer. CAB2 and CAB4 are members of a group of IgG monoclonal antibodies generated against cell surface glycoproteins of the slime mold D. discoideum (35Crandall I.E. Newell P.C. Development. 1989; 107: 87-94PubMed Google Scholar). Reactivity of these antibodies to Dictyostelium cells or cell ghosts was lost or reduced by periodate treatment or digestion with endoglycosidase F, indicating that they were directed against N-linked oligosaccharide epitopes (35Crandall I.E. Newell P.C. Development. 1989; 107: 87-94PubMed Google Scholar). In the present study, they were found to be of the IgG3 subclass. When tested in ELISAs against a panel of glycoproteins with established glycan structures (TableI), both the antibodies reacted with the following: 1) PLA2 which has an oligomannose structure core substituted by fucose residues linked either α1,3 or α1,6 (or is occasionally difucosylated) (37Haslam S.M. Reason A.J. Morris H.R. Dell A. Glycobiology. 1994; 4: 105-111Crossref PubMed Scopus (12) Google Scholar); 2) pFg which has complex biantennary oligosaccharides, core-substituted with fucose linked α1,6 to GlcNAc (33Da Silva M.L.C. Tamura T. McBroom T. Rice K.G. Arch. Biochem. Biophys. 1994; 312: 151-157Crossref PubMed Scopus (28) Google Scholar); and 3) human lactoferrin which has complex biantennary oligosaccharides core-substituted with fucose linked α1,6 to GlcNAc, and additional fucose residues linked α1,3 to GlcNAc on the antennae (38Spik G. Strecker G. Fournet B. Bouquelet S. Montreuil J. Dorland L. van Halbeek H. Vliegenthart J.F.G. Eur. J. Biochem. 1982; 121: 413-419Crossref PubMed Scopus (159) Google Scholar). The antibodies did not bind to core α1,3-fucosylated plant proteins such as HRP (39Kurosaka A. Yano A. Itoh N. Kuroda Y. Nakagawa T. Kawasaki T. J. Biol. Chem. 1991; 266: 4168-4172Abstract Full Text PDF PubMed Google Scholar) or pineapple stem bromelain (40Ishihara H. Takahashi N. Oguri S. Tejima S. J. Biol. Chem. 1979; 254: 10715-10719Abstract Full Text PDF PubMed Google Scholar). The absence of binding to the plant glycoproteins did not result from interference by a β1,2 xylose residue in the core region of these sugar chains, since β-xylosidase digestion of HRP did not increase CAB4 reactivity (Fig. 1). The effectiveness of this digestion is evident from >75% reduction in binding of an affinity purified antibody (see “Experimental Procedures”) against β1,2 xylose (data not shown). CAB4 does not bind to non-core-fucosylated proteins such as 1) bovine fetuin, which has triantennary oligosaccharides, (41Green E.D. Adelt G. Baenziger J.U. Wilson S. Van Halbeek H. J. Biol. Chem. 1988; 263: 18253-18268Abstract Full Text PDF PubMed Google Scholar); or 2) ovalbumin, which has hybrid oligosaccharides, with an intersecting GlcNAc residue (42Tai T. Yamashita K. Ogata-Arakawa M. Koide N. Muramatsu T. Iwashita S. Inoue Y. Kobata A. J. Biol. Chem. 1975; 250: 8569-8575Abstract Full Text PDF PubMed Google Scholar); or 3) human α1-acid glycoprotein, which has complex bi-, tri-, and tetraantennary oligosaccharides, with some fucose residues linked α1,3 to an outer GlcNAc but lacks core fucose substitutions (43Yoshima H. Matsumoto A. Mizuochi T. Kawasaki T. Kobata A. J. Biol. Chem. 1981; 256: 8476-8484Abstract Full Text PDF PubMed Google Scholar). These results indicated that the CAB2 and CAB4 antibodies are probably recognizing core Fucα1,6GlcNAc on N-linked glycans.Table IReactivity of CAB antibodies with some reference glycoproteinsGlycoproteinN-Glycan structureReactive Honey bee venom phospholipase A2Oligomannose glycan, with core Fucα1,3GlcNAcβ/core Fucα1,6GlcNAcβ/difucosylation at the core (37Haslam S.M. Reason A.J. Morris H.R. Dell A. Glycobiology. 1994; 4: 105-111Crossref PubMed Scopus (12) Google Scholar) Porcine fibrinogenComplex biantennary glycan, with core Fucα1,6GlcNAcβ (33Da Silva M.L.C. Tamura T. McBroom T. Rice K.G. Arch. Biochem. Biophys. 1994; 312: 151-157Crossref PubMed Scopus (28) Google Scholar) Human lactoferrinComplex biantennary glycan, with core Fucα1,6GlcNAcβ and outer Fucα1,3GlcNAcβ (38Spik G. Strecker G. Fournet B. Bouquelet S. Montreuil J. Dorland L. van Halbeek H. Vliegenthart J.F.G. Eur. J. Biochem. 1982; 121: 413-419Crossref PubMed Scopus (159) Google Scholar)Non-reactive Horseradish peroxidaseOligomannose glycan, with core Fucα1,3GlcNAcβ and Xylβ1,2Manβ (39Kurosaka A. Yano A. Itoh N. Kuroda Y. Nakagawa T. Kawasaki T. J. Biol. Chem. 1991; 266: 4168-4172Abstract Full Text PDF PubMed Google Scholar) Pineapple stem bromelainOligomannose glycan, with core Fucα1,3GlcNAcβ and Xylβ1,2Manβ (40Ishihara H. Takahashi N. Oguri S. Tejima S. J. Biol. Chem. 1979; 254: 10715-10719Abstract Full Text PDF PubMed Google Scholar) Chicken egg albuminHybrid glycan, intersecting GlcNAc, no core substitution (42Tai T. Yamashita K. Ogata-Arakawa M. Koide N. Muramatsu T. Iwashita S. Inoue Y. Kobata A. J. Biol. Chem. 1975; 250: 8569-8575Abstract Full Text PDF PubMed Google Scholar) Human α1-acid glycoproteinComplex bi-, tri-, and tetraantennary glycans, with outer Fucα1,3GlcNAcβ and no core substitutions (43Yoshima H. Matsumoto A. Mizuochi T. Kawasaki T. Kobata A. J. Biol. Chem. 1981; 256: 8476-8484Abstract Full Text PDF PubMed Google Scholar) Bovine fetuinComplex triantennary glycan, with no core substitutions (41Green E.D. Adelt G. Baenziger J.U. Wilson S. Van Halbeek H. J. Biol. Chem. 1988; 263: 18253-18268Abstract Full Text PDF PubMed Google Scholar) Open table in a new tab Fig. 1 shows CAB4 antibody binding to increasing amounts of pFg, PLA2, HRP, and β-xylosidase-treated HRP measured by ELISA. Linearity was evident up to 100 ng with PLA2 and pFg, with a lower detection limit of 2–5 ng. No reactivity was seen even with 250 ng of either native or dexylosylated HRP. Similar results were seen with CAB2 (not shown). The specificity of both these antibodies was also established by Western blots (Fig. 2). Since the binding pattern for both antibodies is identical, data are shown only for CAB4. A non-relevant monoclonal antibody served as a negative control. The antibodies recognized only core Fucα1,6GlcNAc containing proteins in the blots. Background binding seen with ovalbumin and bromelain was eliminated at higher antibody dilutions (<2 μg/ml). pFg, like other fibrinogens, is composed of three different polypeptides, Aα (69 kDa), Bβ (57 kDa), and γ (51 kDa) chains. The Bβ and γ chains carry core-fucosylated biantennary N-glycans (33Da Silva M.L.C. Tamura T. McBroom T. Rice K.G. Arch. Biochem. Biophys. 1994; 312: 151-157Crossref PubMed Scopus (28) Google Scholar) and are recognized by the CAB antibodies, but the non-glycosylated Aα chain is not. In addition, a higher molecular mass (79 kDa) band is also intensely stained by the antibody. Since fibrinogens are notoriously heterogeneous, this may represent catabolic intermediates of fibrinogen which are often present in plasma or other contaminating binding proteins from commercial pFg. Digestion of PLA2 or pFg with chicken liver α-l-fucosidase, which cleaves fucose in α1→6, →2, →3, →4 linkages, reduces binding >80% (Fig.3). pFg has only core Fucα1,6GlcNAc, but PLA2 also has core Fucα1,3GlcNAc. The digestion did not cleave core α1,3 fucose residues in PLA2 since there was minimal loss of reactivity when probed with the affinity purified anti-Fucα1,3GlcNAc fraction of anti-HRP (Fig. 3,inset). Biantennary glycopeptides containing core-substituted Fucα1,6GlcNAc were generated from pTg (44de Waard P. Koorevaar A. Kamerling J.P. Vliegenthart J.F.G. J. Biol. Chem. 1991; 266: 4237-4243Abstract Full Text PDF PubMed Google Scholar) or pFg. These glycopeptides, or ones from HRP (core Fucα1,3GlcNAc) and ovalbumin (lacking core Fucα1,6GlcNAc), were then compared for their ability to inhibit CAB4 binding to pFg in spectrophotometric or chemiluminescent ELISA. The latter method was adopted when <1 nmol of free inhibitory oligosaccharides was available. As shown in Fig.4, A and B, each assay gave comparable results. In both assays, HRP and ovalbumin glycopeptides did not block antibody binding, but pTg/pFg glycopeptides progressively inhibited CAB4 binding to pFg, again showing the specificity for core Fucα1,6GlcNAc. Biantennary core-fucosylated oligosaccharide from EPO (EPO1, Fig. 4 B) and pFg (not shown) were equally effective inhibitors, showing that GlcNAc-asparagine linkage is probably not required for recognition. To confirm th" @default.
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• W2018172904 cites W1198076339 @default.
• W2018172904 cites W128612890 @default.
• W2018172904 cites W1485462441 @default.
• W2018172904 cites W1504779723 @default.
• W2018172904 cites W1506925756 @default.
• W2018172904 cites W1511697093 @default.
• W2018172904 cites W1515920608 @default.
• W2018172904 cites W1524114939 @default.
• W2018172904 cites W1533411461 @default.
• W2018172904 cites W1550762644 @default.
• W2018172904 cites W1553087448 @default.
• W2018172904 cites W1571588501 @default.
• W2018172904 cites W1577311217 @default.
• W2018172904 cites W1582928646 @default.
• W2018172904 cites W1596459244 @default.
• W2018172904 cites W1602390093 @default.
• W2018172904 cites W1605749013 @default.
• W2018172904 cites W1632006108 @default.
• W2018172904 cites W1949917588 @default.
• W2018172904 cites W1966311210 @default.
• W2018172904 cites W1968578659 @default.
• W2018172904 cites W1971547898 @default.
• W2018172904 cites W1977606211 @default.
• W2018172904 cites W1978986697 @default.
• W2018172904 cites W1979131726 @default.
• W2018172904 cites W1979960661 @default.
• W2018172904 cites W1981370590 @default.
• W2018172904 cites W1983068545 @default.
• W2018172904 cites W1987065820 @default.
• W2018172904 cites W1992357348 @default.
• W2018172904 cites W1996061492 @default.
• W2018172904 cites W1996439275 @default.
• W2018172904 cites W2005616019 @default.
• W2018172904 cites W2007080026 @default.
• W2018172904 cites W2009213314 @default.
• W2018172904 cites W2017496011 @default.
• W2018172904 cites W2019803146 @default.
• W2018172904 cites W2022019156 @default.
• W2018172904 cites W2034465849 @default.
• W2018172904 cites W2034918699 @default.
• W2018172904 cites W2037116242 @default.
• W2018172904 cites W2050081230 @default.
• W2018172904 cites W2054443723 @default.
• W2018172904 cites W2055691615 @default.
• W2018172904 cites W2056083316 @default.
• W2018172904 cites W2056605061 @default.
• W2018172904 cites W2056774453 @default.
• W2018172904 cites W2059331079 @default.
• W2018172904 cites W2070070036 @default.
• W2018172904 cites W2072416953 @default.
• W2018172904 cites W2076413610 @default.
• W2018172904 cites W2081230658 @default.
• W2018172904 cites W2086048344 @default.
• W2018172904 cites W2087881430 @default.
• W2018172904 cites W2088803561 @default.
• W2018172904 cites W2093658667 @default.
• W2018172904 cites W2152708819 @default.
• W2018172904 cites W2186678270 @default.
• W2018172904 cites W4246991801 @default.
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