Abstract
To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.
Highlights
To investigate the factors regulating bthioesynthesis N-acetyllactosamine sequence has been found in complexof poly-N-acetyllactosaminechains containing there- type asparagine-linked oligosaccharides in both soluble and peatingdisaccharide [3GalB1,4GlcNAc@lIin animal membrane-bound animal cell glycoproteins [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15]
Th1' s secell glycoproteins, we have examined the structures quence occurs in keratansulfate (161, in blood group and terminalsequences of these chainsin the comples- antigens [17], and in a number of developmentally regulated typaesparagine-linked oligosaccharides fromthe antigens that areassociated with glycolipidsand glycoproteins mouse lymphoma cell line BW5147
This immobilized lectin interacts with high A portion of the residual glycopeptides eluted in the exaffinity withglycopeptides containing the repeating disaccha-cluded fractions on SephadexG-25 after endo-@-galactosidase ride [3Gal@l,4GlcNAc@l] and with thcoosme plex-type aspar- treatment was desialylated by treatment with mildacid, and agine-linked oligosaccharides containing an outer mannose the desialylated material was reapplied to the SephadexG-25 residue substituted at position C-2 and C-6 by N-acetyllac- column
Summary
1,3Gal; and the high mobility peak is a disaccharide with the Fractions 1-a and I-b were desalted andapplied to a column structure GlcNAc@l,3Gal. of DSA-agarose. This immobilized lectin interacts with high A portion of the residual glycopeptides eluted in the exaffinity withglycopeptides containing the repeating disaccha-cluded fractions on SephadexG-25 after endo-@-galactosidase ride [3Gal@l,4GlcNAc@l] and with thcoosme plex-type aspar- treatment was desialylated by treatment with mildacid, and agine-linked oligosaccharides containing an outer mannose the desialylated material was reapplied to the SephadexG-25 residue substituted at position C-2 and C-6 by N-acetyllac- column. To assess the presence of sialylatedoligosaccharides releasedbyendo-p-galactosidase, a portion of thematerial contained in the Sephadex G-25 void volume (Fig. lA, fractions 14-20) was analyzed directly by descending paper chro-
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