title: Human XTP3-B binds to alpha1-antitrypsin variant null(Hong Kong) via the C-terminal MRH domain in a glycan-dependent manner.
abstract: XTP3-B is a soluble endoplasmic reticulum (ER)-resident protein containing two mannose-6-phosphate receptor homology (MRH) domains in its sequence. XTP3-B interacts with a membrane-associated ubiquitin ligase complex, and, therefore, is thought to participate in ER-associated degradation (ERAD). In this study, the recombinant human XTP3-B fused with IgG-Fc (XTP3-B-Fc), XTP3-B without an N-terminal MRH domain fused with IgG-Fc (XTP3-BDelta1-Fc), or XTP3-B without a C-terminal MRH domain fused with IgG-Fc (XTP3-BDelta2-Fc) were prepared. XTP3-B-Fc and XTP3-BDelta1-Fc bound to Lec1 cells but not to CHO, Lec2, or Lec8 cells, while XTP3-BDelta2-Fc did not bind to any of these cells. The binding of XTP3-B-Fc and XTP3-BDelta1-Fc to Lec1 cells was abrogated by treatment of the cells with endo-beta-N-acetylglucosaminidase H, Manalpha1,6Man or Manalpha1,6(Manalpha1,3)Manalpha1,6(Manalpha1,3)Man, or by substitution of Arg428 or Tyr457 in the C-terminal MRH domain with alanine. Arg428 and Tyr457 are homologous to amino acids that mediate glycan binding by the cation-dependent mannose-6-phosphate receptor. An immunoprecipitation experiment using lysates of cells co-expressing wild-type alpha1-antitrypsin (AT), alpha1-antitrypsin variant null(Hong Kong) (AT(NHK)), and FLAG-tagged XTP3-B, or its mutants, demonstrated that AT(NHK), but not AT, specifically co-precipitated with XTP3-B and XTP3-BDelta1. The glycan-binding-deficient XTP3-BDelta2 did not bind either AT or AT(NHK). These results suggest that XTP3-B specifically binds to AT(NHK), which is a well-known substrate of ERAD, via a C-terminal MRH domain in a glycan-dependent manner.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yamaguchi
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.forename: Naoki
authors.initials: N
authors.lastname: Matsumoto
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D014644:Genetic Variation; D006801:Humans; D037102:Lectins / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: C531192:ERLEC1 protein, human; D037102:Lectins; D011134:Polysaccharides; D000515:alpha 1-Antitrypsin