Glycosylation of human adiponectin affects its conformation and stability.
The collagenous region of adiponectin is glycosylated in vitro with glucosylgalactosyl moieties on four conserved lysines. We investigated the glycosylation of human adiponectin in vivo. Sugar vicinyl hydroxides on adiponectin were oxidized with 10 or 1 mM metaperiodate, and the result analyzed by two-dimensional electrophoresis and immunoblotting. Only 10 mM metaperiodate caused significant changes in electrophoretic mobility and an altered susceptibility to proteinase K digestion. Such treatment also increased the susceptibility of hexamers and high molecular weight (HMW) isoforms to dissociation by SDS. By contrast, untreated low molecular weight (LMW) isoforms were readily dissociated by low concentrations of SDS. Reduced HMW isoforms were able to partially reassemble following the removal of dithiothreitol, and this process was unaffected by metaperiodate. The presence of sialic acid was detected by Maackia amurensis Lectin II blotting, and by oxidation with 1 mM metaperiodate, followed by detection with Emerald Green 300 fluorescent dye. Quantitation of sugars on affinity-purified adiponectin from nine human plasmas showed that dimers of HMW isoforms contained a 1.3-fold greater amount of total sugar than LMW isoforms. However, both contained similar amounts of sialic acid. We conclude that glucosylgalactosyl residues contribute to the conformation of HMW human plasma adiponectin. In addition, the HMW isoform contains greater amounts of glucosylgalactosyl residues than the LMW isoform, and these sugars are important in determining its stability in vivo.
Peake
Philip W
PW
Division of Medicine, Prince of Wales Hospital, Randwick, New South Wales 2031, Australia. p.peake@unsw.edu.au
Hughes
Jaquelyne T
JT
Shen
Yvonne
Y
Charlesworth
John A
JA
D052242:Adiponectin / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000273:Adipose Tissue / Q000378:metabolism*; D004355:Drug Stability; D004586:Electrophoresis; D015180:Electrophoresis, Gel, Two-Dimensional; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D037102:Lectins; D008297:Male; D008970:Molecular Weight; D011487:Protein Conformation
D052242:Adiponectin; D037102:Lectins; D005947:Glucose; D005690:Galactose