Publications

We present a general technique for fractionating cell-derived asparagine-linked oligosaccharides on the basis of oligosaccharide structure. This procedure has been applied to the study of [2-3H]mannose-labeled mouse lymphoma cells (BW5147). The fractionation scheme involves serial chromatography on concanavalin A-Sepharose, pea lectin-Sepharose, and leukoagglutinating phytohemagglutinin-agarose. Approximately 85% of the labeled glycopeptides was retained on one or more of the affinity columns. The various fractions eluted from the columns contain relatively pure populations of glycopeptides which were used for structural analysis. The recovery of the glycopeptides was quantitative. The procedure was used to estimate the overall spectrum of Asn-linked oligosaccharides synthesized by the lymphoma cell line. We conclude that serial lectin-agarose affinity chromatography is a rapid, sensitive, and specific technique for fractionating and analyzing Asn-linked oligosaccharides. A general fractionation scheme employing additional lectins is presented.

The carbohydrate binding specificities of the leukoagglutinating phytohemagglutinin (L-PHA) and erythroagglutinating phytohemagglutinin (E-PHA) lectins of the red kidney bean, Phaseolus vulgaris, have been investigated by lectin-agarose affinity chromatography of Asn-linked oligosaccharides. High affinity binding to E-PHA-agarose occurs only with biantennary glycopeptides containing 2 outer galactose residues and a residue of N-acetylglucosamine linked beta 1,4 to the beta-linked mannose residue in the core. This species is not retarded on L-PHA-agarose. In contrast, tri- and tetraanternnary glycopeptides containing outer galactose residues and an alpha-linked mannose residue substituted at positions C-2 and C-6 are specifically retarded on L-PHA-agarose. Triantennary glycopeptides containing outer galactose residues and an alpha-linked mannose residue substituted at positions C-2 and C-4 are not retarded on L-PHA-agarose. Additionally, the presence of outer sialic acid residues or a core fucose residue does not influence the behavior of complex glycopeptides on either of these lectin-agarose conjugates. This ability of E-PHA and L-PHA to discriminate between Asn-linked oligosaccharides with various branching patterns can be utilized in the fractionation of these glycopeptides (see paper following).

Murine Ia antigens consist of two glycosylated polypeptide chains, the alpha chain and the beta chain. We have used lectin affinity chromatography to confirm previous work in our laboratory that three distinct, differentially glycosylated I-Ak alpha chains (alpha 1, alpha 2, and alpha 3) exist and to compare the carbohydrate of the alpha chain with that of the beta chain. Glycopeptides derived from pronase digestion of [3H]mannose-labeled I-Ak alpha 1, alpha 2, alpha 3, and beta chains were sequentially passed over columns of immobilized concanavalin A, Lens culinaris lectin, phytohemagglutinin-E, and phytohemagglutinin-L in a prescribed manner to generate a lectin affinity profile, which, in turn, allowed assignment of a minimal oligosaccharide structure for each glycopeptide studied. The lectin affinity profile for each chain was unique. The alpha 1, alpha 2, and beta chains each possess complex-type N-linked oligosaccharides, although the branching pattern and specific sugar residues found on each differ. The alpha 3 chain, on the other hand, possesses predominantly high mannose or hybrid-type N-linked oligosaccharides. Lectin affinity analysis of glycopeptides derived from pronase digestion of high pressure liquid chromatography-isolated tryptic-chymotryptic fragments from alpha 2 and alpha 3 and tryptic fragments from beta revealed that specific minimal oligosaccharide structures were associated with particular fragments. In addition, although tryptic-chymotryptic peptide maps of alpha 2 and alpha 3 were similar, alpha 2 fragments bear predominantly complex-type N-linked oligosaccharides, whereas homologous alpha 3 fragments bear high mannose or hybrid-type N-linked oligosaccharides. Possible explanations of the oligosaccharide heterogeneity are discussed.

Upon incubation with uridine diphosphate-[14C]glucuronic acid, membrane fractions from adult and phenobarbital-induced embryonic liver synthesize a single glucuronide, which is soluble in chloroform:methanol (2:1). The compound is completely hydrolyzed and glucuronic acid released by either mild acid or beta-glucuronidase, whereas mild base hydrolysis results in a mixture of glucuronic acid and glucuronic acid-1,2-cyclic phosphate. These data and the behavior of the lipid-linked glucuronide on DEAE-cellulose chromatography indicate that the compound contains a monophosphate diester of glucuronic acid, which is beta-linked to a lipid. The synthesis of the lipid-linked glucuronide in uninduced normal embryonic liver is very low (5-15 pmol product/mg/5 min) at all developmental ages up to hatching, but the introduction of phenobarbital into the air space of a 9-10-day-old embryo causes a premature increase of activity (75-150 pmol products/mg/5 min) within 7 days. The glucuronyltransferase in adult and induced embryonic liver has a Km for UDPGlcUA of 0.17 x 10(-3) M and a broad pH optimum between pH 6 and 7. Glucuronic acid is released from the lipid-linked glucuronide by a beta-glucuronidase in liver that is active at neutral pH and is not inhibited by saccharolactone. This glycosidase activity appears, therefore, to be distinct from the previously characterized lysosomal beta-glucuronidase. Fractionation of adult chicken liver membranes by differential centrifugation indicates that over 70% of the glucuronyltransferase is associated with the nuclear and mitochondrial fractions. The endogenous beta-glucuronidase capable of hydrolyzing the lipid-linked glucuronide was not separated from the glucuronyl-transferase activity during fractionation. The data available suggests that the lipid-linked glucuronide is involved directly in the generation of free glucuronic acid for further metabolism.

A galactosyltransferase that transfers galactose from UDPgalactose to asialoagalacto fetuin or N-acetylglucosamine was partly purified from two commerical preparations of fetuin and its kinetic properties were characterized. Several other preparations of fetuin were also found to contain galactosyltransferase activity.