title: The interactions of calreticulin with immunoglobulin G and immunoglobulin Y.
abstract: Calreticulin is a chaperone of the endoplasmic reticulum (ER) assisting proteins in achieving the correctly folded structure. Details of the binding specificity of calreticulin are still a matter of debate. Calreticulin has been described as an oligosaccharide-binding chaperone but data are also accumulating in support of calreticulin as a polypeptide binding chaperone. In contrast to mammalian immunoglobulin G (IgG), which has complex type N-glycans, chicken immunoglobulin Y (IgY) possesses a monoglucosylated high mannose N-linked glycan, which is a ligand for calreticulin. Here, we have used solid and solution-phase assays to analyze the in vitro binding of calreticulin, purified from human placenta, to human IgG and chicken IgY in order to compare the interactions. In addition, peptides from the respective immunoglobulins were included to further probe the binding specificity of calreticulin. The experiments demonstrate the ability of calreticulin to bind to denatured forms of both IgG and IgY regardless of the glycosylation state of the proteins. Furthermore, calreticulin exhibits binding to peptides (glycosylated and non-glycosylated) derived from trypsin digestion of both immunoglobulins. Additionally, calreticulin peptide binding was examined with synthetic peptides covering the IgG Cγ2 domain demonstrating interaction with approximately half the peptides. Our results show that the dominant binding activity of calreticulin in vitro is toward the polypeptide moieties of IgG and IgY even in the presence of the monoglucosylated high mannose N-linked oligosaccharide on IgY.
authors: Department of Clinical Biochemistry and Immunology, Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark.
authors: Karen Mai
authors: KM
authors: Møllegaard
authors: Karen
authors: K
authors: Duus
authors: Sofie Dietz
authors: SD
authors: Træholt
authors: Morten
authors: M
authors: Thaysen-Andersen
authors: Yan
authors: Y
authors: Liu
authors: Angelina S
authors: AS
authors: Palma
authors: Ten
authors: T
authors: Feizi
authors: Paul R
authors: PR
authors: Hansen
authors: Peter
authors: P
authors: Højrup
authors: Gunnar
authors: G
authors: Houen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D037282:Calreticulin / Q000737:chemistry / Q000378:metabolism*; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D040081:Protein Array Analysis; D011485:Protein Binding; D011489:Protein Denaturation; D014357:Trypsin / Q000378:metabolism
chemical_list: D037282:Calreticulin; C016512:IgY; D007074:Immunoglobulin G; D007136:Immunoglobulins; D009844:Oligosaccharides; D010455:Peptides; D014357:Trypsin