Biosynthesis of N‐Glycolylneuraminic Acid in Porcine Submandibular Glands

Abstract

1. The morphology of porcine submandibular gland tissue is demonstrated by light and electron microscopy.2. Subcellular particles from porcine submandibular glands were prepared by a procedure adapted to the presence of viscous mucus, and were fractionated on continuous and discontinuous sucrose gradients.3. Six particulate fractions were obtained by discontinuous gradient centrifugation. These were identified by electron microscopy, marker enzymes and nucleic acid content and were designated as follows: (1) plasma membranes, (2) smooth membranes and light Golgi membranes, (3) heavy Golgi membranes, (4) membrane mixture and mitochondria, ( 5 ) mitochondria and (6) membrane mixture and mucin residues. The bulk of rough membranes and cell nuclei was found in the 3000 x g sediment before fractionation.4. Sialyltransferase and N-acetylneuraminate monooxygenase showed highest activities in the Golgi membrane fractions representing 45 % and 36 x, respectively, of total tissue activity. The specific activities of these membrane-bound enzymes were respectively 13 and 10 times higher when compared with the homogenate. 5. The acylneuraminic acid/protein ratio was lowest in the Golgi membranes when compared with the other particulate fractions and the cytosol glycoprotein. The molar N-acetylneuraminic acid/N-glycolylneuraminic acid ratio was highest in the two Golgi membrane fractions (average 45: 55) and in the pool of free acylneuraminic acids (41 : 59), and lowest in the cytosol glycoprotein (10 : 90).6. Approximately 2.6 o/;l of the total acylneuraminic acids occurring in porcine submandibular glands are freely diffusible, 80 % of them representing free N-acetylneuraminic acid and N-glycolylneuraminic acid in a molar ratio of about 4: 6 and 20 % representing cytidylyl-glycosides of N-acetylneuraminic acid and N-glycolylneuraminic acid in a ratio of 6 : 4.7. In radioactive labelling experiments with surviving slices of porcine submandibular glands the highest specific radioactivities were found in the pool of free acylneuraminic acids, followed by Nacetylneuraminic acid and N-glycolylneuraminic acid bound to the Golgi membranes. N-Acetylneuraminic acid and N-glycolylneuraminic acid of the cytosol glycoproteins have lowest turnover rates.8. Based on these experiments a model is presented describing two pathways of N-acetylneuramink acid modification in the course of glycoprotein biosynthesis. At least 40 x of N-acetylneuraminic acid is hydroxylated before its transfer onto Golgi membranes and linkage to growing glycoprotein molecules and about 50 thereafter. This results in a molar N-acetylneuraminic acid/N-glycolylneuraminic acid relationship of 1 : 9 in the secreted glycoprotein. It is assumed that free N-acetylneuramink acid is modified by the soluble cytosol monooxygenase whereas the glycoprotein-bound N-acetylneuraminic acid is hydroxylated by the corresponding membrane-bound enzyme.

Porcine submaxillary mucin contains α2→3‐ and α2→6‐linked N‐acetyl‐ and N‐glycolyl‐neuraminic acid

“…For instance, Rearick et al [q have reported the characterization of porcine submaxillary P-galactoside a2 +3-sialyltransferase, to form the sequence NeuAca(2-t 3)-Galfl(1+3)GalNAc, although this structural element had not been reported in porcine submaxillary mucin (PSM). In addition, a study by Sherblom and Dahlin [lo] showing that both NeuAc and NeuGc were present in tumour cell glycoproteins, along with the report of Buscher et al [l 11 on the biosynthesis of NeuGc in porcine submaxillary glands and the considerable controversy which has surrounded the oligosaccharide structures of PSM [3 -51 prompted a further investigation.…”

Inhibition of γ‐glutamyltranspeptidase by treatment of intact lymphocytes with periodate

“…When cells are oxidized with periodate and treated with [3H] borohydride, 4 labeled glycoproteins can be seen on subsequent SDS gel electrophoresis which are not labeled if lymph node cells are oxidized by neuraminidase plus galactose oxidase; these are in addition to -20 components labeled in common after either method of oxidation. These 'periodate specific' glycoproteins may contain 4-Oacylated sialic acids which are resistant to neuraminidase but susceptible to periodate [28,29]. Two such glycoproteins on lymph node cells have M,-values of -28 000 and 30 000 which are near that of the small, catalytic subunit of rat y-glutamyltranspeptidase [21]; one of these is sensitive to papain.…”

Evidence for a free N-acetylneuraminic acid-hydroxylating enzyme in pig mandibular gland soluble fraction

“…This recent discovery goes against one made earlier showing free NeuSAc to be the authentic substrate for the pig mandibular gland enzyme [14]. Due to our interest in the expression of Neu5Gc in malignancy, we have embarked on the study of Neu5Gc biosynthesis in pig mandibular gland.…”