Identification and characterization of major proteins carrying ABO blood group antigens in the human kidney.
BACKGROUND
It is generally admitted that ABO(H) blood group antigens are linked to lipids and proteins. Although glycolipids carrying ABO antigens have been well characterized in human kidneys, glycoproteins carrying ABO antigens are largely unknown, and their molecular properties remain to be elucidated.


METHODS
All the blood group A antigen-linked proteins in human kidney could be solubilized and captured on immobilized Helix pomatia lectin that recognizes A antigens. These proteins were separated on SDS-PAGE gels. The gel pieces containing protein bands immunoreactive with anti-A antibody were excised, in-gel digested with trypsin, and analyzed by nanoLC tandem mass spectrometer. Protein candidates that carry ABO antigens were confirmed by immunoprecipitation and double-labeled immunofluorescense microscopy.


RESULTS
All the glycoproteins carrying ABO antigens were found to be Asn-linked glycoproteins, and presented as multiple bands on SDS-PAGE with molecular masses ranging from 60 to 270 kDa. The protein bands were subjected for mass spectrometric analysis, which identified 121 distinct proteins with high confidence. Of the identified proteins, 55 N-glycosylated, membrane proteins were selected as glycoprotein candidates that carry ABO antigens. Among them, most abundantly expressed proteins as estimated by the number of peptide matches in the MS spectrometric analysis, such as platelet endothelial cell adhesion molecule 1, plasmalemmal vesicle-associated protein, and von Willebrand factor, were further characterized.


CONCLUSIONS
Several glycoproteins were identified that represented major glycoproteins carrying ABO antigens in the human kidney, which exhibited distinct features in localization to most of vascular endothelial cells.
Tasaki
Masayuki
M
Department of Structural Pathology, Institute of Nephrology, Niigata Graduate School of Medical and Dental Sciences, Niigata, Japan.
Yoshida
Yutaka
Y
Miyamoto
Masahito
M
Nameta
Masaaki
M
Cuellar
Lino M
LM
Xu
Bo
B
Zhang
Ying
Y
Yaoita
Eishin
E
Nakagawa
Yuki
Y
Saito
Kazuhide
K
Yamamoto
Tadashi
T
Takahashi
Kota
K
D000017:ABO Blood-Group System / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001789:Blood Group Antigens / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004730:Endothelium, Vascular / Q000276:immunology; D058851:GPI-Linked Proteins; D006017:Glycolipids / Q000032:analysis; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification; D006372:Helix, Snails / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000166:cytology / Q000276:immunology* / Q000648:ultrastructure; D037102:Lectins / Q000276:immunology; D016253:Microscopy, Immunoelectron; D009363:Neoplasm Proteins / Q000032:analysis; D012079:Renal Circulation / Q000276:immunology; D014841:von Willebrand Factor / Q000032:analysis
D000017:ABO Blood-Group System; D015703:Antigens, CD; D001789:Blood Group Antigens; C120523:CD109 protein, human; D058851:GPI-Linked Proteins; D006017:Glycolipids; D006023:Glycoproteins; C038095:Helix lectin; D037102:Lectins; D009363:Neoplasm Proteins; D014841:von Willebrand Factor