title: Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris.
abstract: Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.
authors.affiliation: Analytical Development, GlycoFi, Inc., Merck & Co., 21 Lafayette Street, Suite 200, Lebanon, New Hampshire 03766, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Cukan
authors.forename: Daniel
authors.initials: D
authors.lastname: Hopkins
authors.forename: Irina
authors.initials: I
authors.lastname: Burnina
authors.forename: Michelle
authors.initials: M
authors.lastname: Button
authors.forename: Erin
authors.initials: E
authors.lastname: Giaccone
authors.forename: Nga Rewa
authors.initials: NR
authors.lastname: Houston-Cummings
authors.forename: Youwei
authors.initials: Y
authors.lastname: Jiang
authors.forename: Fang
authors.initials: F
authors.lastname: Li
authors.forename: Muralidhar
authors.initials: M
authors.lastname: Mallem
authors.forename: Teresa
authors.initials: T
authors.lastname: Mitchell
authors.forename: Renée
authors.initials: R
authors.lastname: Moore
authors.forename: Adam
authors.initials: A
authors.lastname: Nylen
authors.forename: Bianka
authors.initials: B
authors.lastname: Prinz
authors.forename: Sandra
authors.initials: S
authors.lastname: Rios
authors.forename: Nathan
authors.initials: N
authors.lastname: Sharkey
authors.forename: Dongxing
authors.initials: D
authors.lastname: Zha
authors.forename: Stephen
authors.initials: S
authors.lastname: Hamilton
authors.forename: Huijuan
authors.initials: H
authors.lastname: Li
authors.forename: Terrance A
authors.initials: TA
authors.lastname: Stadheim
mesh_terms: D000818:Animals; D016466:CHO Cells; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D006224:Cricetinae; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D037181:Lectins, C-Type / Q000378:metabolism*; D008358:Mannose / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D015202:Protein Engineering; D011956:Receptors, Cell Surface / Q000378:metabolism*; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
chemical_list: D015815:Cell Adhesion Molecules; D006023:Glycoproteins; D007074:Immunoglobulin G; D037181:Lectins, C-Type; D008358:Mannose; D011134:Polysaccharides; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin