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ABSTRACT: vWF (von Willebrand factor) is a key component for maintenance of normal haemostasis, acting as the carrier protein of the coagulant Factor VIII and mediating platelet adhesion at sites of vascular injury. There is ample evidence that vWF glycan moieties are crucial determinants of its expression and function. Of particular clinical interest, ABH antigens influence vWF plasma levels according to the blood group of individuals, although the molecular mechanism underlying this phenomenon remains incompletely understood. The present paper reports analyses of the human plasma vWF N-glycan population using advanced MS. Glycomics analyses revealed approximately 100 distinct N-glycan compositions and identified a variety of structural features, including lactosaminic extensions, ABH antigens and sulfated antennae, as well as bisecting and terminal GlcNAc residues. We estimate that some 300 N-glycan structures are carried by human vWF. Glycoproteomics analyses mapped ten of the consensus sites known to carry N-glycans. Glycan populations were found to be distinct, although many structural features were shared across all sites. Notably, the H antigen is not restricted to particular N-glycosylation sites. Also, the Asn2635 site, previously designated as unoccupied, was found to be highly glycosylated. The delineation of such varied glycan populations in conjunction with current models explaining vWF activity will facilitate research aimed at providing a better understanding of the influence of glycosylation on vWF function.
Biochemical Journal 07/2012; 447(2):217-28. · 4.90 Impact Factor
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ABSTRACT: The potential for protein-engineered biotherapeutics is enormous, but pharmacokinetic modulation is a major challenge. Manipulating pharmacokinetics, biodistribution, and bioavailability of small peptide/protein units such as antibody fragments is a major pharmaceutical ambition, illustrated by the many chemical conjugation and recombinant fusion approaches being developed. We describe a recombinant approach that leads to successful incorporation of polysialic acid, PSA for the first time, onto a therapeutically valuable protein. This was achieved by protein engineering of the PSA carrier domain of NCAM onto single-chain Fv antibody fragments (one directed against noninternalizing carcinoembryonic antigen-CEA and one against internalizing human epidermal growth factor receptor-2-HER2). This created novel polysialylated antibody fragments with desired pharmacokinetics. Production was achieved in human embryonic kidney cells engineered to express human polysialyltransferase, and the recombinant, glycosylated product was successfully fractionated by ion-exchange chromatography. Polysialylation was verified by glycosidase digestion and mass spectrometry, which showed the correct glycan structures and PSA chain length similar to that of native NCAM. Binding was demonstrated by ELISA and surface plasmon resonance and on live cells by flow cytometry and confocal immunofluorescence. Unexpectedly, polysialylation inhibited receptor-mediated endocytosis of the anti-HER2 scFv. Recombinant polysialylation led to an estimated 3-fold increase in hydrodynamic radius, comparable to PEGylation, leading to an almost 30-fold increase in blood half-life and a similar increase in blood exposure. This increase in bioavailability led to a 12-fold increase in tumor uptake by 24 h. In summary, recombinant polysialylation of antibody fragments in our system is a novel and feasible approach applicable for pharmacokinetic modulation, and may have wider applications.
Bioconjugate Chemistry 06/2012; 23(8):1524-33. · 4.93 Impact Factor
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M Tony Hollingsworth,
Gerald W Hart,
James C Paulson,
Elizabeth Stansell, Kevin Canis,
I-Cheuh Huang,
Maria Panico,
Howard Morris,
Stuart Haslam,
Michael Farzan, [......],
Kazunori Hamamura,
Takenosuke Yoshida,
Kaoru Akita,
Tetsuya Okajima,
Keiko Furukawa,
Takeshi Urano,
Koichi Furukawa,
L Renee Ruhaak,
Suzanne Miyamoto,
Carlito B Lebrilla
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ABSTRACT: Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.
Glycobiology 11/2011; 21(11):1454-531. · 3.58 Impact Factor
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ABSTRACT: Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.
Blood 06/2011; 117(24):6685-93. · 9.90 Impact Factor
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ABSTRACT: Although stretches of serine and threonine are sometimes sites for O-linked carbohydrate attachment, specific sequence and structural determinants for O-linked attachment remain ill defined. The gp120 envelope protein of SIVmac239 contains a serine-threonine-rich stretch of amino acids at positions 128 to 139. Here we show that lectin protein from jackfruit seed (jacalin), which binds to non- and monosialylated core 1 O-linked carbohydrate, potently inhibited the replication of SIVmac239. Selection of a jacalin-resistant SIVmac239 variant population resulted in virus with specific substitutions within amino acids 128 to 139. Cloned simian immunodeficiency virus (SIV) variants with substitutions in the 128-to-139 region had infectivities equivalent to, or within 1 log unit of, that of SIVmac239 and were resistant to the inhibitory effects of jacalin. Characterization of the SIVmac239 gp120 O-linked glycome showed the presence of core 1 and core 2 O-linked carbohydrate; a 128-to-139-substituted variant gp120 from jacalin-resistant SIV lacked O-linked carbohydrate. Unlike that of SIVmac239, the replication of HIV-1 strain NL4-3 was resistant to inhibition by jacalin. Purified gp120s from four SIVmac and SIVsm strains bound jacalin strongly in an enzyme-linked immunosorbent assay, while nine different HIV-1 gp120s, two SIVcpz gp120s, and 128-to-139-substituted SIVmac239 gp120 did not bind jacalin. The ability or inability to bind jacalin thus correlated with the presence of the serine-threonine-rich stretch in the SIVmac and SIVsm gp120s and the absence of such stretches in the SIVcpz and HIV-1 gp120s. Consistent with sequence predictions, two HIV-2 gp120s bound jacalin, while one did not. These data demonstrate the presence of non- and monosialylated core 1 O-linked carbohydrate on the gp120s of SIVmac and SIVsm and the lack of these modifications on HIV-1 and SIVcpz gp120s.
Journal of Virology 10/2010; 85(1):582-95. · 5.40 Impact Factor
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Chih-Jen Wei,
Ling Xu,
Wing-Pui Kong,
Wei Shi, Kevin Canis,
James Stevens,
Zhi-Yong Yang,
Anne Dell,
Stuart M Haslam,
Ian A Wilson,
Gary J Nabel
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ABSTRACT: Although the human transmission of avian H5N1 virus remains low, the prevalence of this highly pathogenic infection in avian species underscores the need for a preventive vaccine that can be made without eggs. Here, we systematically analyze various forms of recombinant hemagglutinin (HA) protein for their potential efficacy as vaccines. Monomeric, trimeric, and oligomeric H5N1 HA proteins were expressed and purified from either insect or mammalian cells. The immunogenicity of different recombinant HA proteins was evaluated by measuring the neutralizing antibody response. Neutralizing antibodies to H5N1 HA were readily generated in mice immunized with the recombinant HA proteins, but they varied in potency depending on their multimeric nature and cell source. Among the HA proteins, a high-molecular-weight oligomer elicited the strongest antibody response, followed by the trimer; the monomer showed minimal efficacy. The coexpression of another viral surface protein, neuraminidase, did not affect the immunogenicity of the HA oligomer, as expected from the immunogenicity of trimers produced from insect cells. As anticipated, HA expressed in mammalian cells without NA retained the terminal sialic acid residues and failed to bind alpha2,3-linked sialic acid receptors. Taken together, these results suggest that recombinant HA proteins as individual or oligomeric trimers can elicit potent neutralizing antibody responses to avian H5N1 influenza viruses.
Journal of Virology 08/2008; 82(13):6200-8. · 5.40 Impact Factor
