Novel hyperglycosylated weak gelatin-binding fibronectin from human fetal placenta. Fractionation of a high poly(N-acetyllactosamine) fragment by tomato lectin affinity chromatography.
A novel hyperglycosylated fraction of human term fetal placental fibronectin was detected by long-term affinity binding to gelatin-Sepharose. An 18-h batch-wise gelatin-binding step was necessary to obtain a very low-affinity binding fraction, characterized by especially high N-acetylglucosamine and galactose content, and diffuse, poorly stained Coomassie bands on SDS/polyacrylamide electrophoretograms. The presence of a high proportion of long 7-10-kDa poly(N-acetyllactosamine)-containing N-linked carbohydrate chains was confirmed by their gel permeation behavior, susceptibility to endo-beta-galactosidase and by methylation analysis. Our previous results suggest that 4.5-7-kDa poly(N-acetyllactosamine) structures reduce the binding of fibronectin and its chymotryptic Ala260-Trp599 subdomain GB44 to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041-4045]. Based on a gradient of urea used to dissociate gelatin-bound GB44, in the present study, fractions containing the novel 7-10-kDa carbohydrates showed significantly weaker binding to gelatin. Weak gelatin-binding characteristics of this novel hyperglycosylated fraction suggest that extended poly(N-acetyllactosamine) N-linked chains can significantly weaken heterotropic binding functions of fetal glycoproteins. The combined properties of weak Coomassie staining and weak gelatin binding have caused the novel hyperglycosylated fibronectin to be overlooked in previous investigations.
Zhu
B C
BC
Department of Biochemistry, Louisiana State University, Baton Rouge 70803.
Laine
R A
RA
D000328:Adult; D002241:Carbohydrates / Q000032:analysis; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D005260:Female; D005353:Fibronectins / Q000302:isolation & purification* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D005780:Gelatin / Q000378:metabolism*; D017127:Glycation End Products, Advanced; D006801:Humans; D037102:Lectins; D010446:Peptide Fragments / Q000302:isolation & purification; D010920:Placenta / Q000032:analysis*; D011134:Polysaccharides / Q000302:isolation & purification*; D011247:Pregnancy; D014508:Urea; D001616:beta-Galactosidase; D000093844:Glycated Proteins
D002241:Carbohydrates; D002352:Carrier Proteins; D002918:Chymotrypsin; D005353:Fibronectins; D005780:Gelatin; D017127:Glycation End Products, Advanced; D037102:Lectins; D010446:Peptide Fragments; D011134:Polysaccharides; D014508:Urea; D001616:beta-Galactosidase; C033135:gelatin binding protein; C045099:glycated fibronectin; D000093844:Glycated Proteins; C037199:poly-N-acetyllactosamine