title: Sugar-binding proteins from fish: selection of high affinity "lambodies" that recognize biomedically relevant glycans.
abstract: Glycan-binding proteins are important for a wide variety of basic research and clinical applications, but proteins with high affinity and selectivity for carbohydrates are difficult to obtain. Here we describe a facile and cost-effective strategy to generate monoclonal lamprey antibodies, called lambodies, that target glycan determinants. We screened a library of yeast surface-displayed (YSD) lamprey variable lymphocyte receptors (VLR) for clones that can selectively bind various biomedically important glycotopes. These glycoconjugates included tumor-associated carbohydrate antigens (Tn and TFα), Lewis antigens (LeA and LeX), N-glycolylneuraminic acid, targets of broadly neutralizing HIV antibodies (poly-Man9 and the HIV gp120), and the glycoproteins asialo-ovine submaxillary mucin (aOSM) and asialo-human glycophorin A (aGPA). We isolated clones that bind each of these targets in a glycan-dependent manner and with very strong binding constants, for example, 6.2 nM for Man9 and 44.7 nM for gp120, determined by surface plasmon resonance (SPR). One particular lambody, VLRB.aGPA.23, was shown by glycan array analysis to be selective for the blood group H type 3 trisaccharide (BG-H3, Fucα1-2Galβ1-3GalNAcα), aGPA, and TFα (Galβ1-3GalNAcα), with affinity constants of 0.2, 1, and 8 nM, respectively. In human tissue microarrays this lambody selectively detected cancer-associated carbohydrate antigens in 14 different types of cancers. It stained 27% of non-small cell lung cancer (NSCLC) samples in a pattern that correlated with poor patient survival. Lambodies with exquisite affinity and selectivity for glycans may find myriad uses in glycobiology and biomedical research.
authors.affiliation: Institute of Marine and Environmental Technology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Columbus Center Suite 236, 701 East Pratt St, Baltimore, Maryland 21202, United States.
authors.forename: Xia
authors.initials: X
authors.lastname: Hong
authors.forename: Mark Z
authors.initials: MZ
authors.lastname: Ma
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Gildersleeve
authors.forename: Sudipa
authors.initials: S
authors.lastname: Chowdhury
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Barchi
authors.forename: Roy A
authors.initials: RA
authors.lastname: Mariuzza
authors.forename: Michael B
authors.initials: MB
authors.lastname: Murphy
authors.forename: Li
authors.initials: L
authors.lastname: Mao
authors.forename: Zeev
authors.initials: Z
authors.lastname: Pancer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D000951:Antigens, Neoplasm / Q000187:drug effects; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D006801:Humans; D007798:Lampreys; D008175:Lung Neoplasms / Q000175:diagnosis / Q000276:immunology; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D012015:Reference Standards
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D011134:Polysaccharides