Ceruloplasmin carries the anionic glycan oligo/poly alpha2,8 deaminoneuraminic acid

Oligo/poly alpha2,8 deaminoneuraminic acid (KDN), a unique posttranslational protein modification, was found on megalin and a not yet characterized 150 kDa glycoprotein. We purified this glycoprotein from rat testis and identified it as ceruloplasmin. Furthermore, immunoprecipitated ceruloplasmin from rat thymus, ovary, blood serum, and postnatal day 2 but not adult lung and brain was immunoreactive for oligo/poly alpha2,8 KDN. The immunoreactivity for oligo/poly alpha2,8 KDN on purified serum ceruloplasmin was abolished by N-glycosidase F treatment but not by beta-elimination, indicating that it is present on N-glycosidically linked oligosaccharides. However, the copper binding activity of ceruloplasmin was independent of the presence of the anionic glycan. By immunohistochemistry, ceruloplasmin was detectable in histologically defined regions in rat ovary, thymus, and spleen. Likewise, by RT-PCR, ceruloplasmin expression was found in various non-hepatic rat tissues and showed a developmentally regulated pattern. Thus, ceruloplasmin, in addition to megalin, represents a glycoprotein carrying oligo/poly alpha2,8 KDN.     

Analytical methods for identifying and quantitating deamidated sialic acid (2-keto-3-deoxy-D-glycero-D-galactonononic acid) and alpha 2----8-linked poly(oligo)nonulosonate residues in glycoconjugates.

In 1986 we reported the natural occurrence of deaminated neuraminic acid (2-keto-3-deoxy-D-glycero-D-galactonononic acid, KDN) in fish egg glycoprotein. Subsequently, we have shown that many types of sialic acid as well as KDN occur in polymeric chains, poly(oligo)-Sia and poly(oligo)KDN in nature. In this study we demonstrate that the conventional colorimetric and gas-liquid chromatographic methods used in the analysis of sialic acid can be applied to analysis of these new nonulosonate and poly(oligo)nonulosonates. We report that the thiobarbituric acid reaction can be used to analyze both free and bound KDN, but gives lower extinction values when applied to poly(oligo)KDN without prior hydrolysis. Further, the published hydrolytic and/or methanolytic procedures are suitable to release the terminal sialic acid residues, but are not appropriate for quantitative release of the nonulosonic acids from poly(oligo)nonulosonates. A new gas-liquid chromatographic procedure for the identification-quantitation of nonulosonates in poly(oligo)meric forms is described.     

Synthesis,characterization, and preliminary biological study of glycoconjugates of poly(styrene-co-maleic acid)

Three derivatives of the biocompatible polymer poly(styrene-co-maleic anhydride) (SMA) were obtained with 1-amino-1-deoxy-beta-D-galactose, 1-amino-1-deoxy-beta-D-glucose, and 1-amino-1-deoxy-beta-D-lactose, respectively. The amino sugars were chemically conjugated via formation of an amide bond between the anomeric amino group of the sugar residue and the anhydride of the copolymer, giving the corresponding glycoconjugate derivatives. Colorimetric assay of the unreacted amino groups and elemental analysis were used to determine the degree of substitution. About 56%, 54%, and 94% of the available anhydride groups reacted to give galactosyl-amide (SMA-Gal), glucosyl-amide (SMA-Gluc), and lactosyl-amide (SMA-Lac) branched polymers, respectively. The synthesized glycopolymers were characterized by Fourier transform infrared spectroscopy, gel permeation chromatography, circular dichroism, and UV and fluorescence spectroscopy. The release of glucosylamine from the glucosyl-amide branched polymer, by basic hydrolysis, was monitored by high-performance anion-exchange chromatography and by capillary electrophoresis, providing for an additional check of the degree of substitution of this specific polymer derivative. Biological activity tests showed that both SMA-Gal and SMA-Lac allow adhesion of HepG2 hepatic cells about five times larger than that of hydrolyzed, underivatized SMA.     

Expression of oligo/polyα2,8-linked deaminoneuraminic acid and megalin during kidney development and maturation: mutually exclusive distribution with polyα2,8-linked N-acetylneuraminic acid of N-CAM

The expression of homopolymers of α2,8-linked deaminoneuraminic acid (oligo/polyα2,8-KDN) and of megalin during rat kidney development was investigated using immunocytochemistry and immunoblotting, and compared to homopolymers of α2,8-linked N-acetylneuraminic acid (polyα2,8-Neu5Ac) of the neural cell adhesion molecule (N-CAM). Both, oligo/polyα2,8-KDN and megalin were found in early proximal tubules of embryonic day 18 kidneys. In addition, megalin, but not oligo/polyα2,8-KDN, was detectable in late S-shaped bodies and early capillary loop stages. Until postnatal day 7, oligo/polyα2,8-KDN and megalin immunoreactivity was present, not only in convoluted but also in straight proximal tubules, and then restricted to the convoluted part as in adult kidney. Immunoblotting revealed increasing megalin expression until postnatal week 3 of kidney development, when the level corresponded to adult kidney. Combined immunoprecipitation/immunoblot analyses showed a steady level of oligo/polyα2,8-KDN on megalin throughout development. This was in striking contrast to the expression of polyα2,8-Neu5Ac and N-CAM, which was highest in early embryonic kidney, undetectable in kidneys of 3-week-old rats, and mutually exclusive with oligo/polyα2,8-KDN in its distribution. These findings demonstrated the coincidence of oligo/polyα2,8-KDN and megalin expression and the first appearance of proximal tubules, and revealed the high degree of specialization of the biosynthetic machinery for protein polysialylation in kidney. Accepted: 9 July 1999     

A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.     

Cloning, expression, and characterization of sialic acid synthases

The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.     

Synthesis and characterization of poly(dimer acid-brassylic acid) copolymer and poly(dimer acid-pentadecandioic acid) copolymer

Poly(dimer acid-brassylic acid) [P(DA-BA)] copolymers and poly(dimer acid-pentadecandioic acid) [P(DA-PA)] copolymers were prepared by melt polycondensation of the corresponding mixed anhydride prepolymers. The copolymers were characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle x-ray powder-diffraction, and thermal gravimetric analysis (TGA). In vitro studies show that all the copolymers are degradable in phosphate buffer at 37 degrees C, and leaving an oily dimer acid residue after hydrolysis for the copolymer with high content of dimer acid. The release profiles of hydrophilic model drug, ciprofloxcin hydrochloride, from the copolymers, follow first-order release kinetics. All the preliminary results suggested that the copolymer might be potentially used as drug delivery devices.     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

Structural and functional aspects of oligo(uridylic acid)-containing and poly(adenylic acid)-containing ribonucleic acids from rat liver

Uridylate tracts were released from rat liver mRNA by nuclease digestion and terminally-labeled invitro with ³²P using polynucleotide kinase. The pattern of fragments released from A⁺ and U⁺ mRNAs were the same as judged by electrophoresis on urea-polyacrylamide gels. The bulk of the fragments were in the size range 20–40 residues, but larger components (up to 70 residues in length) could also be seen. The ability of U⁺ and A⁺ mRNAs to direct the synthesis of proteins in a rabbit reticulocyte cell-free system was evaluated. The translation products were resolved by two-dimensional polyacrylamide gel electrophoresis. A comparison of the proteins made by the two classes of RNA showed that the U⁺ mRNA fraction represents a subset of A⁺ mRNA species, although the proportions of the protein products were quite different and several proteins were found to be unique to the U⁺ mRNA class.     

The histochemical application of dansylhydrazine as a fluorescent labeling reagent for sialic acid in cellular glycoconjugates.

A new method for the fluorescent staining of stalic acid-containing glycoconjugates in fixed tissues is described. The procedure uses mild periodate oxidation, followed by condensation with dansylhydrazine and reduction of the hydrazones to hydrazines. The specificity of the reaction for sialic acid is tested on model glycoconjugates. The procedure gives superior resolution in comparison to the standard periodate Schiff procedure for cellular carbohydrates.     

Relative amounts of sialic acid and fucose of amniotic fluid glycoconjugates in relation to pregnancy age

The present knowledge concerning the glycan structures and role of glycoconjugates derived from amniotic fluid is fragmentary and mainly focuses on the individual glycoproteins. The question has arisen as whether the general glycosylation pattern of amniotic fluid glycoconjugates can change with the progression of a normal pregnancy. In the present work we have described the dynamic, quantitative alterations in relative amounts of sialic acid and fucose linked by a variety of anomeric linkages to subterminal oligosaccharide structures of amniotic fluid glycoconjugates in relation to pregnancy age. The analysis was performed in the following groups of amniotic fluids derived from normal pregnancy by lectin dotting method: “2nd trimester” (14–19 weeks), “3rd trimester” (29–37 weeks), “perinatal period” (38–40 weeks) , “delivery at term” (39–41 weeks) and “post date pregnancy” (41–43 weeks). In the “3rd trimester” the amniotic fluid glycoconjugates contained higher relative amounts of glycans terminated by α2-6-linked sialic acid (p < 0.00002) and by α1-6 innermost fucose (p < 0.000001) than those in the 2nd trimester. In contrast, they showed the lower relative amount of fucose linked α1-3 (p < 0.02). At the perinatal period the relative amount of α2-6-linked sialic acid increased (p < 0.03), and it then decreased during delivery (p < 0.02) to the level found in the “3rd trimester” group. In the post date pregnancy all parameters studied increased. The sialyl- and fucosyl-glycotopes of the amniotic fluid glycoconjugates may play an critical role in growth and tissue remodeling of the foetus, as well as may might reflect maturation of a foetus. Additionally, a determination of the glycotope expressions might be helpful in prenatal diagnosis as predictor factors for well being of mother and child.     

In situ copolyesters containing poly(L-lactide) and poly(hydroxyalkanoate) units

In situ copolyesters containing polylactide (PLA) and polyhydroxyalkanoate (PHA) segments were obtained via ring-opening polymerization of L-lactide using PHA as a macroinitiator with stannous octoate as catalyst. Incorporation of PHA (20 wt %) into PLA affords a novel copolymer with Mn values ranging from 25 to 50 KDa and low polydispersities of 1.8-2.3. DSC analysis of the copolymer indicates well-defined crystallization and melting transitions different from the homopolymers and corresponding blend. The polymers were characterized by FT-IR, GPC, DSC, optical microscopy, NMR, and TGA. The results show successful reactivity of PHA as a macroinitiator for the ring-opening polymerization of lactide.     

Photoresponsive polypeptides: Photochromism and conformation of poly (L-glutamic acid) containing spiropyran units

Spirobenzopyran units were bound to the side chains of poly (L -glutamic acid) and partially methylated poly(L -glutamate)s. The modified polymers were found to exhibit “reverse photochromism” in hexafluoro-2-propanol (HFP), so the samples kept in the dark were characterized by an intense absorption band in the visible range of the spectrum, which was completely erased upon exposure to sunlight or irradiation at 500–550 nm. The CD spectra showed that the macromolecules adopted a random coil conformation in the dark, whereas the bleached solutions after exposure to light displayed the typical CD pattern of the α-helix. The back reaction in the dark was accompanied by the progressive decrease of the helix content and recovery of the original disordered conformation. The photoinduced conformational changes resulted in large and reversible viscosity variations. When spiropyran side chains were converted to “spiropyran salts” of trifluoroacetic acid, the system was still photochromic, but the macromolecules were disordered both in the dark and light conditions. However, when appropriate amounts of methanol were added as a cosolvent to the HFP solutions, the system responded to light, giving reversible variations of the α-helix content. Irradiation at appropriate solvent compositions allowed modulation of the extent of the photoresponse. © 1993 John Wiley & Sons, Inc.     

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949–56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326–31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19–48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60–81 pmol/mg protein in spleen and lung and 6.9–18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs.