Frederic A. Troy

Frequent Occurrence of Pre-existing α2→8-Linked Disialic and Oligosialic Acids with Chain Lengths Up to 7 Sia Residues in Mammalian Brain Glycoproteins PREVALENCE REVEALED BY HIGHLY SENSITIVE CHEMICAL METHODS AND ANTI-DI-, OLIGO-, AND POLY-Sia ANTIBODIES SPECIFIC FOR DEFINED CHAIN LENGTHS

The pre-existence of α2→8-linked disialic acid (di-Sia) and oligosialic acid (oligo-Sia) structures with up to 7 Sia residues was shown to occur on a large number of brain glycoproteins, including neural cell adhesion molecules (N-CAMs), by two highly sensitive chemical methods (Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1998) Anal. Biochem. 261, 191–197; Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1999) Anal. Biochem. 266, 102–109). This unexpected finding was also confirmed using a newly developed antibody prepared using a copolymer of α2→8-linked N-acetylneuraminylp-vinylbenzylamide and acrylamide as an immunogen and known antibodies whose immunospecificities were determined to be di- and oligo-Sia residues with defined chain lengths. The major significance of the new finding that di- and oligo-Sia chains exist on a large number of brain glycoproteins is 2-fold. First, it reveals a surprising diversity in the number and M r of proteins distinct from N-CAM that are covalently modified by these short sialyl glycotopes. Second, it suggests that synthesis of di- and/or oligo-Sia units may be catalyzed by α2→8-sialyltransferase(s) that are distinct from the known polysialyltransferases, STX and PST, which are partially responsible for polysialylation of N-CAM.

Degree of Polymerization (DP) of Polysialic Acid (PolySia) on Neural Cell Adhesion Molecules (N-CAMs) DEVELOPMENT AND APPLICATION OF A NEW STRATEGY TO ACCURATELY DETERMINE THE DP OF polySIA CHAINS ON N-CAMS

α2,8-Linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal α2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-β-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs ∼50–60 or less, a subpopulation of chains with DPs ∼150 to ∼180 and extending to DP ∼400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-β-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

Polysialic Acid, a Glycan with Highly Restricted Expression, Is Found on Human and Murine Leukocytes and Modulates Immune Responses

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV−/− mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.

Biosynthesis of KDN (2-Keto-3-deoxy-d-glycero-d-galacto-nononic acid) IDENTIFICATION AND CHARACTERIZATION OF A KDN-9-PHOSPHATE SYNTHETASE ACTIVITY FROM TROUT TESTIS

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-d-glycero-d-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP → Man-6-P + ADP; 2) Man-6-P + PEP → KDN-9-P + Pi; 3) KDN-9-P → KDN + Pi. Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to formd-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn2+. N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation ofN-acetyl-d-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199–27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.

Nuclear magnetic resonance studies of polyisoprenols in model membranes

2H-NMR investigation of polyisoprenols (PIs) in model membranes has revealed information about their motions, relative order, and locale within the membrane. Initial 2H-NMR studies of the organization of the shorter chain homologues geraniol (C10), farnesol (C15), and solanesol (C45) were carried out by incorporating 2H-acetyl esters of the alcohol or the di-perdeuterome-thylated derivatives of the omega-labeled prenols into multilamellar phosphatidylcholine (PC) vesicles. 2H-NMR powder patterns interpretable in terms of quadrupole splittings and spin-lattice relaxation times were obtained. Similar experiments have now been carried out with the labeled free alcohol, acetyl ester, and phosphate ester of dolichol (C95) and undecaprenol (C55). 2H-NMR results show that the head and tail 2H-labeled sites of C55 and C95 exhibit a fast motion isotropic signal only; no slower motion anisotropy, as exhibited by the short chain PIs, was observed. These data suggest that C55 and C95 either have substantially different (faster) motions and/or conformations relative to the shorter chain PIs within the membrane, and that the longer PIs alter the membrane host packing matrix. This conclusion was supported by 31P-NMR studies of C55 and C95 derivatives in PC and PE/PC membranes, which showed new pronounced spectral changes relative to the results obtained with the shorter chain PIs. These spectral changes indicate that undecaprenol and dolichol derivatives appear to induce a non-bilayer (isotropic) organization of phospholipid molecules in PE/PC (2:1) vesicles. The possible physiological consequences of this perturbation remains to be determined.

Sialobiology and the Polysialic Acid Glycotope Occurrence, Structure, Function, Synthesis, and Glycopathology

Polysialic acids (polySia)* are a structurally diverse family of linear carbohydrate chains that consist of N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) residues, usually joined internally by α2,8-, α2,9-, or alternating α2,8-/α2,9-ketosidic linkages. 3-Deoxy-D-glycero-D-galacto-2- nonulosonic acid (KDN) is a unique deaminated form of Sia, and polyKDN chains share many properties in common with polySia (Table I). The finding of poly(Neu5Ac), poly(Neu5Gc), poly(Neu5Ac,Neu5Gc), poly(KDN) chains and their partially O-acetylated and O-lactylated forms in salmonid fish egg glycoproteins demonstrates the natural occurrence of multiple forms of these unique sugar chains.

Serologic analysis of antigen-specific reactivity in patients with systemic candidiasis

Antibody responses to candidal polypeptides and mannans were studied in patients with systemic candidiasis, candiduria, and other fungal and bacterial infections, and in healthy laboratory personnel to determine the diagnostic value of these immunologic responses. When tested by immunoblot analysis, sera from 15 patients with systemic candidiasis frequently contained antibodies to three antigens: 15 of 15 sera from patients with invasive disease reacted to a molecular species having a molecular weight (Mr of 90-200 kd, 13 of 15 reacted with a 45-kd polypeptide, and 12 of 15 reacted with a 17-kd polypeptide. Lesser reactivity was observed in 11 of 15 sera with a 28-kd candidal antigen and in 9 of 15 to a 57-kd candidal antigen. Quantitation of antibody titers against the 45-kd candidal polypeptide demonstrated much higher immunoreactivity in patients with systemic candidiasis than in patients with superficial candidal infections, bacterial infections, other systemic mycoses, and healthy individuals. Antimannan antibody titers were measured by an enzyme-linked immunosorbent assay (ELISA) and these titers were also higher in patients with systemic candidiasis than in patients in the other categories. These differences, however, were less than those observed with the anti-45-kd polypeptide antibody. Therefore, the ability to detect systemic candidiasis is improved by testing sera for immunoreactivity to polypeptide and to mannan antigens from Candida albicans. Detection of polypeptide antibodies improves the serodiagnosis of systemic candidiasis.

Induction, Localization, and Purification of a Novel Sialidase, Deaminoneuraminidase (KDNase), from Sphingobacterium multivorum

Recently, we reported the discovery of a new type of sialidase, KDNase, which specifically hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), but not N-acylneuraminyl linkages. We now report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum. Growth of S. multivorum in the presence of KDN-containing oligosaccharide alditols, KDNα23Galβ13GalNAcα13[KDNα2 (8KDNα2)6]GalNAcol, as a sole carbon source induced KDNase SM activity 15-40-fold, compared with growth in the absence of inducer. KDN, Neu5Ac, or Neu5Ac oligomers were ineffective as inducers. The enzyme was released from the periplasm of induced cells by cold osmotic shock and purified 700-fold to homogeneity. The specific activity of the pure enzyme was 82,100 units/mg of protein. KDNase SM activity resided in a single polypeptide chain with an estimated molecular weight of approximately 47,500. Enzyme activity was maximal at near neutral pH. The availability of pure KDNase will now make it possible to study the structure and functional role of KDN-glycoconjugates and to determine the molecular mechanism whereby the enzyme can discriminate between KDN and N-acylneuraminic acid.

Identification of polysialic acids in glycoconjugates

Publisher Summary This chapter provides a description of the strategies and methods used for detecting, analyzing, and modifying polysialic acids (polySia) and their derivatives. Several highly specific and sensitive anti-polySia and anti-2-keto-3-deoxy- D -glycero- D -galacto-nononic acid (KDN) antibodies have been developed to identify α -2,8-linked polySia, (KDN)G M3 , or oligoKDN expression in prokaryotic and eukaryotic cells. Endo N is the key enzyme for confirming the presence of polySia. It is specific for hydrolyzing oligo- or polySia residues in sources as distinct as bacteria and human brain. The K1F Endo N has been widely used in both immunodetection procedures and polysialyltransferase reactions to prove the existence of α -2,8-polySia by catalyzing its depolymerization. The chapter presents immunochemical methods for rapidly detecting α -2,8-linked oligo/polySia chains in cell or tissue homogenates that allow this epitope to be readily detected by Western blot or rocket immunoelectrophoresis, using the H.46 antibody. Similar procedures using monoclonal antibodies, enzyme-linked immunnosorbent assays (ELISAs), and immunocytochemistry are also described.

Rapid separation of oligomers of polysialic acid by high-performance liquid chromatography

A rapid procedure utilizing high-performance liquid chromatography was developed for the separation of homooligomers of sialic acid (N-acetylneuraminic acid). The method utilizes the anion exchanger Mono-Q HR 5/5 and can resolve sialyl oligomers with degrees of polymerization (DP) from 2 to 20 in 25 min. Previous methods required 1 to 9 days. Recoveries are quantitative and the method can be used either analytically to analyze the enzymatic digestion products of polysialic acid or semipreparatively to prepare sialyl oligomers of defined length. The method is potentially useful for analyzing other anionic oligosaccharides.