title: Expression of glycoproteins bearing complex human-like glycans with galactose terminal in Hansenula polymorpha.
abstract: Glycoproteins derived from Hansenula polymorpha can not be used for therapeutic purposes due to their high-mannose type asparagine-linked (N-linked) glycans, which result in immune reactions and poor pharmacokinetic behaviors in human body. Previously, we reported that the trimannosyl core N-linked glycans (Man(3)GlcNAc(2)) intermediate can be generated in endoplasmic reticulum in HpALG3 and HpALG11 double-mutant H. polymorpha. Here, we describe the further modification of the glycosylation pathway in this double-defect strain to express glycoproteins with complex human-like glycans. After eliminating the impact of HpOCH1, three glycosyltransferases were introduced into this triple-mutant strain. When human β-1,2-N-acetylglucosaminyltransferase I (hGnTI) was efficiently targeted in early Golgi, more than 95 % glycans attached to the glycoproteins were added one N-acetylglucosamine (GlcNAc). With subsequently introduction of rat β-1,2-N-acetylglucosaminyltransferase II (rGnTII) and human β-1,4-galactosyltransferase I (hGalTI), several glycoengineered strains can produce glycoproteins bearing glycans with terminal N-acetylglucosamine or galactose. The expression of glycoproteins with glycan Gal(2)GlcNAc(2)Man(3)GlcNAc(2) represents a significant step toward the ability to express fully humanized glycoproteins in H. polymorpha. Furthermore, several shake-flask and bioreactor fermentation experiments indicated that, although the cells do display a reduction in growth rate, the glycoengineered strains are still suitable for high-density fermentation.
authors.affiliation: Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Hao-Lei
authors.initials: HL
authors.lastname: Song
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.forename: Bing-Sheng
authors.initials: BS
authors.lastname: Qiu
mesh_terms: D000818:Animals; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005690:Galactose / Q000737:chemistry*; D005818:Genetic Engineering; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D016695:Glycosyltransferases / Q000378:metabolism; D006801:Humans; D010843:Pichia / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D051381:Rats
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D016695:Glycosyltransferases; D005690:Galactose