External labelling of glycoproteins from first-trimester human placental microvilli

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The brush-border glycoproteins of first-trimester human placentas were investigated by using two external labelling techniques: (1) sequential digestion with neuraminidase and galactose oxidase, followed by reduction with NaB3H4, which 3H-labels terminal galactose and galactosaminase residues; and (2) sequential treatment with periodate and NaB3H4, which 3H-terminal sialic acid residues. The labelling procedures were performed on intact tissue so that the results would more closely approximate the topography of the bursh border in vivo. The microvilli were isolated, subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, and the [3H]glycoproteins detected by fluorography. Densitometer scans of the fluorograms of the [3H]galactoproteins showed that, under reducing conditions, 90% of the protein-associated radioactivity was incorporated into two glycoproteins. The major [3H]galactoprotein of early placental microvilli had an estimated molecular mass of 92kDa (desialylated) and migrated as a diffuse band. A minor 180kDa glycoprotein was less consistently labelled. No change in the apparent molecular mass of either component was detected in the absence of β-mercaptoethanol, suggesting that the 180kDa component was not a dimer of the 92kDa glycoprotein. The remaining 10% of the radioactivity was equally distributed among several minor membrane components. Densitometer scans of the fluorograms of the [3H]sialoproteins showed that, under either reducing on non-reducing conditions, 90% of the 3H was preferentially incorporated into the 92-110kDa region of the gel. Although no distinct bands were visible, the higher-molecular-mass region of this area was always most heavily labelled. A minor 180kDa glycoprotein was also 3H-labelled. The pattern of brush-border [3H]glycoproteins from first-trimester placentas differed markedly from that of term placental microvilli and from placental fibroblast plasma membranes that were 3H-labelled by identical external labelling techniques. These results indicate that: (1) the glycoprotein determinants of brush-border topography change during pregnancy: (2) within the placenta, the major 92kDa (desialylated) determinant, which has not been previously described, is unique to the trophoblastic cells.

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Biochemical Journal

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