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Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products.

ABSTRACT: High-pressure liquid chromatography was used to identify the sialo-oligosaccharide products obtained after sialylation of [14C]Gal-GalNAc-protein in vitro by an ovine submaxillary-gland microsomal fraction. Among other products, two isomeric trisaccharides could be identified. NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol and Gal beta 1 leads to 3-(NeuAc alpha 2 leads to 6)GalNAcol respectively, indicating that ovine submaxillary gland contains two sialyltransferases acting on mucin-type acceptors, a beta-galactoside alpha 2 leads to 3 sialyltransferase and a N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. This conclusion was fully supported by methylation analysis of the two trisaccharide products.

SUBMITTER: Bergh ML

PROVIDER: S-EPMC1163658 | biostudies-other | 1982 Feb

REPOSITORIES: biostudies-other

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Project description:Ca(2+)-independent lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) is a member of the phospholipase A(2) superfamily with a distinguishing characteristic of high specificity for oxidatively modified sn-2 fatty acid residues in phospholipids that has been especially well characterized for peroxidized species of phosphatidylcholines (PC). The ability of Lp-PLA(2) to hydrolyze peroxidized species of phosphatidylserine (PS), acting as a recognition signal for clearance of apoptotic cells by professional phagocytes, as well as the products of the reaction has not been investigated. We performed liquid chromatography-electrospray ionization mass spectrometry-based structural characterization of oxygenated, hydrolyzed molecular species of PS-containing linoleic acid in either the sn-2 position (C(18:0)/C(18:2)) or in both sn-1 and sn-2 positions (C(18:2)/C(18:2)), formed in the cytochrome c- and H(2)O(2)-driven enzymatic oxidation reaction. Cytochrome c has been chosen as a catalyst of peroxidation reactions because of its likely involvement in PS oxidation in apoptotic cells. We found that Lp-PLA(2) catalyzed the hydrolysis of both nontruncated and truncated (oxidatively fragmented) species of oxidized PS species, albeit with different efficiencies, and performed detailed characterization of the major reaction products: oxygenated derivatives of linoleic acid as well as nonoxygenated and oxygenated species of lyso-PS. Among linoleic acid products, derivatives oxygenated at the C(9) position, including 9-hydroxyoctadecadienoic acid (9-HODE), a potent ligand of G protein-coupled receptor G2A, were the most abundant. Computer modeling of interactions of Lp-PLA(2) with different PS-oxidized species indicated that they are able to bind in the proximity (<5 Å) of Ser273 and His351 of the catalytic triad. For 9-hydroxy and 9-hydroperoxy derivatives of oxidized PS, the sn-2 ester bond was positioned very close (<3 Å) to the Ser273 residue, a nucleophile directly attacking the sn-2 bond, thus favoring the hydrolysis reaction. We suggest that oxidatively modified free fatty acids and lyso-PS species generated by Lp-PLA(2) may represent important signals facilitating and regulating the execution of apoptotic and phagocytosis programs essential for the control of inflammation.

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Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products.

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