D H Van den Eijnden

Specificity in the enzymic transfer of sialic acid to the oligosaccharide branches of BI- and triantennary glycopeptides of α1-acid glycoprotein

Abstract Partial in vitro sialylation of biantennary and triantennary glycopeptides of α1-acid glycoprotein using colostrum β-galactosideα(2→6) sialyltransferase followed by high resolution 1H-NMR spectroscopic analysis of the isolated products enabled the assignment of the Galβ(1→4)GlcNAcβ(1→2)Man α(1→3) Man branch as the most preferred substrate site for sialic acid attachment. The Galβ(1→4)GlcNAcβ(1→2)Man α(1→6) Man branch appeared to be much less preferred and the Galβ(1→4)GlcNAcβ(1→4)Manα(1→3)Man sequence of triantennary structures was of intermediate preference for the sialyltransferase. The specificity of the β-galactoside α(2→6) sialyltransferase is thus shown to extend to structural features beyond the terminal N -acetyllactosamine units on the oligosaccharide chains of serum glycoproteins.

ISOLATION AND CHARACTERIZATION OF A SOLUBLE GLUCOSE‐CONTAINING SIALOGLYCOPROTEIN FROM THE CORTICAL GREY MATTER OF CALF BRAIN

A sialoglycoprotein has been isolated from the cortical grey matter of calf brain after homogenization in 0.32 M‐sucrose or in 0.15 M‐NaCl. The sialoglycoprotein is present in the supernatant obtained after centrifugation at 100,000 g for 60 min. It is designated GP‐350 on account of its elution with 350 mM‐NaCl on a DEAE‐cellulose column. From DEAE‐cellulose chromatography it is evident that compounds comparable to GP‐350 occur in the brain of calf and sheep, whereas they seem to be absent in calf liver and kidney. After purification, with polyacrylamide gel electrophoresis only one band can be shown both at pH 8.9 and 7.5. GP‐350 consists of about 83 percent of protein and about 17 per cent of carbohydrate. The polypeptide core has an acidic character: amino acid analysis gives 26 per cent for glutamic acid plus aspartic acid and their amides, with a ratio of acidic to basic amino acids of 3.3. The carbohydrate moiety contains 2.4% sialic acid, 5.5 % hexosamine and 9.4% hexose. It is remarkable that this brain sialoglycoprotein comprises 4% glucose. Care was taken to prevent contamination with glucose‐containing materials during the purification procedure of GP‐350. The complete absence of other glucose‐containing compounds which occur in brain, Le. glycogen and gangliosides, was demonstrated. GP‐350 accounts for at least 3 per cent of the total saline‐extractable protein and about 20 per cent of the total saline‐extractable protein‐bound sialic acid of the cortical grey matter of calf brain. These percentages correspond to 390 pg of protein and to 14 μg of sialic acid per g wet weight. GP‐350 remains soluble when the pH is brought to 3.9 or when ethanol is added to 70 % (v/v).

Sialoglycoproteins, gangliosides and related enzymes in developing rat brain.

It has been shown, that a similarity in regional distribution exists between sialoglycoproteins and gangliosides in bovine brain [l] . In rat brain as well as in bovine brain most of the gangliosides and the sialoglycoproteins are found in the mitochondrial and the microsomal fractions [2-4,6] . Most evidence points to a localization in dendritic and axonal membranes as well as in their synaptic connections [2,4-6, 71. Dekirmenjian and Brunngraber [4] showed the distribution of gangliosides and sialoglycoproteins to be different after subfractionation of a fraction enriched in axones and nerve-ending particles. We supposed that information about the distribution of these sialo-compounds could also be obtained from the developmental pattern. Therefore the levels of gangliosides and sialoglycoproteins during development were determined. For comparison the activities of sialidase (EC 3.2.1.18) CMP-NANA * synthetase, p-galactosidase (EC 3.2.1.13) and P-galactosaminidase were measured. Our data show that the rate of most rapid increase of both gangliosides and sialoglycoproteins is between the 4th and the 18th day. These changes more closely parallel the maximal rate of axonal than of dendritic increase [8] .

THE SUBCELLULAR LOCALIZATION OF CYTIDINE 5′-MONOPHOSPHO-N-ACETYLNEURAMINIC ACID SYNTHETASE IN CALF BRAIN

Abstract— The subcellular distribution of cytidine 5′‐monophospho‐N‐acetylneuraminic acid synthetase, which is a key enzyme involved in the biosynthesis of sialo‐glycoproteins and gangliosides, was studied in the frontal grey cortex and the corpus callosum of the calf brain. It appeared that the enzyme was highly concentrated in nuclear fractions, which were shown to be relatively pure as evaluated from morphological, enzymic and chemical data. The possibility that the nuclear localization of the enzyme was due to an artifact, as the result of adsorption onto the nuclear membrane or absorption into the nucleus during the homogenization of the tissue, was ruled out completely and so it appeared that cytidine 5′‐monophospho‐N‐acetylneuraminic acid synthetase, at least for the greater part, is a true nuclear enzyme. The subnuclear localization of the enzyme was shown to be in the nuclear sap. Both neuronal and glial cell nuclei contained the enzyme, which makes it very likely that biosynthesis of sialo‐glycoproteins and/or gangliosides occurs in neurons as well as in glial cells. The significance of the nuclear localization of the enzyme for the biosynthesis of sialic acid containing macromolecules and the possible regulatory role played by the nucleus in this process are discussed.

Deletion of two exons from the Lymnaea stagnalis ß164-N-acetylglucosaminyltransferase gene elevated the kinietic efficiency of the encoded enzyme for both UDP-sugar donor and acceptor substrates

Lymnaea stagnalisUDP-GlcNAc:GlcNAcβ-R β1→4-N-acetylglucosaminyltransferase (β4-GlcNAcT) is an enzyme with structural similarity to mammalian UDP-Gal:GlcNAcβ-R β1→4-galactosyltransferase (β4-GalT). Here, we report that also the exon organization of the genes encoding these enzymes is very similar. The β4-GlcNAcT gene (12.5 kilobase pairs, spanning 10 exons) contains four exons, encompassing sequences that are absent in the β4-GalT gene. Two of these exons (exons 7 and 8) show a high sequence similarity to part of the preceding exon (exon 6), suggesting that they have originated by exon duplication. The exon in the β4-GalT gene, corresponding to β4-GlcNAcT exon 6, encodes a region that has been proposed to be involved in the binding of UDP-Gal. The question therefore arose, whether the repeating sequences encoded by exon 7 and 8 of the β4-GlcNAcT gene would determine the specificity of the enzyme for UDP-GlcNAc, or for the less preferred UDP-GalNAc. It was found that deletion of only the sequence encoded by exon 8 resulted in a completely inactive enzyme. By contrast, deletion of the amino acid residues encoded by exons 7 and 8 resulted in an enzyme with an elevated kinetic efficiency for both UDP-sugar donors, as well as for its acceptor substrates. These results suggest that at least part of the donor and acceptor binding domains of the β4-GlcNAcT are structurally linked and that the region encompassing the insertion contributes to acceptor recognition as well as to UDP-sugar binding and specificity.

THE REGIONAL DISTRIBUTION OF CYTIDINE 5′‐MONOPHOSPHO‐N‐ACETYL‐NEURAMINIC ACID SYNTHETASE IN CALF BRAIN

—The enzyme cytidine 5′‐monophospho‐N‐acetylneuraminic acid synthetase was studied in different parts of the calf brain. Characterization of partial purified enzyme preparations from cortical grey matter and corpus callosum by means of pH optima and Km values, showed the enzyme of grey and white brain areas to be identical. Unexpectedly the regional differences of the enzyme activities per g wet tissue and per mg protein were very slight. From the presence of the enzyme in pure white brain areas, which are known to be poor in neuronal perikarya, and the fact that the enzyme is localized in the cell nucleus, we concluded that cytidine 5′‐monophospho‐N‐acetylneuraminic acid is produced in glia cell nuclei and that it is very likely that biosynthesis of sialo‐glycoproteins and/or ganglio‐sides occurs within glia cells.

S6.3 Expression of soluble rcombinant αl,3-galactosyltransferase in insect cells

Proteoglycans were prepared from human synovial fluid, blood and articular cartilage using a new method based on precipitation with Alcian Blue. Proteoglycans in a solution of 4 M guanidine-HC1 can be specifically precipitated with the cationic dye Alcian Blue at low pH and in the presence of detergent. Neither proteins or unsulphated glycosaminoglycans are precipitated. The proteoglycan-Alcian Blue complexes are dissociated in a mixture of guanidine-HC1 and propanol. The proteoglycans are precipitated by increasing the propanol concentration and the dye is used for quantitation and the proteoglycans can be separated by electrophoresis in gels of pure agarose, using a discontinuous buffer system that allows stacking of the sample. Large proteoglycans from articular cartilage were separated into three populations by electrophoresis. The synovial fluid contained three large size proteoglycan fragments but no small fragments or free glycosaminoglycans were found. In blood plasma only very small proteoglycans were detected. Electron microscopy of the proteoglycans from synovial fluid revealed fragments from both the C-terminal and N-terminal domains of the large proteoglycans. SDS-PAGE patterns of enzymatically deglycosylated proteoglycans extracted from the cartilage and from synovial fluid were compared. In both cases two major core protein sizes were found with several intermediate sizes, but the synovial proteoglycan fragments were smaller than the cartilage molecules.