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A Varki

Publications and source records attributed to A Varki.

At least 73 records · Page 4Linked to original sources
PubMed1996

Calcium-dependent oligonucleotide antagonists specific for L-selectin.

The selectins are calcium-dependent C-type lectins that recognize complex anionic carbohydrate ligands, initiating many cell-cell interactions in the vascular system. Selectin blockade shows therapeutic promise in a variety of inflammatory and postischemic pathologies. However, the available oligosaccharide ligand mimetics have low affinities and show cross-reaction among the three selectins, precluding efficient and specific blockade. The SELEX (systematic evolution of ligands by exponential enrichment) process uses combinatorial chemistry and in vitro selection to yield high affinity oligonucleotides with unexpected binding specificities. Nuclease-stabilized randomized oligonucleotides subjected to SELEX against recombinant L-selectin yielded calcium-dependent antagonists with approximately 10(5) higher affinity than the conventional oligosaccharide ligand sialyl LewisX. Most of the isolated ligands shared a common consensus sequence. Unlike sialyl LewisX, these antagonists show little binding to E- or P-selectin. Moreover, they show calcium-dependent binding to native L-selectin on peripheral blood lymphocytes and block L-selectin-dependent interactions with the natural ligands on high endothelial venules.

Base Sequence↗
PubMed1996

Molecular cloning and characterization of lysosomal sialic acid O-acetylesterase.

O-Acetylation and de-O-acetylation of sialic acids have been implicated in the regulation of a variety of biological phenomena, including endogenous lectin recognition, tumor antigenicity, virus binding, and complement activation. Applying a strategy designed to identify genes preferentially expressed in active sites of embryonic hematopoiesis, we isolated a novel cDNA from the pluripotent hematopoietic cell line FDCPmixA4 whose open reading frame contained sequences homologous to peptide fragments of a lysosomal sialic acid O-acetylesterase (Lse) previously purified from rat liver, but with no evident similarity to endoplasmic reticulum-derived acetylesterases. The expressed Lse protein exhibits sialic-acid O-acetylesterase activity that is not attributable to a typical serine esterase active site. lse expression is spatially and temporally restricted during embryogenesis, and its mRNA levels correlate with differences in O-acetylesterase activity described in adult tissues and blood cell types. Using interspecific backcross analysis, we further mapped the lse gene to the central region of mouse chromosome 9. This constitutes the first report on the molecular cloning of a sialic acid-specific O-acetylesterase in vertebrates and suggests novel roles for the 9-O-acetyl modification of sialic acids during the development and differentiation of mammalian organisms.

Acetylesterase↗
PubMed1996

Uptake and incorporation of an epitope-tagged sialic acid donor into intact rat liver Golgi compartments. Functional localization of sialyltransferase overlaps with beta-galactosyltransferase but not with sialic acid O-acetyltransferase.

The transfer of sialic acids (Sia) from CMP-sialic acid (CMP-Sia) to N-linked sugar chains is thought to occur as a final step in their biosynthesis in the trans portion of the Golgi apparatus. In some cell types such Sia residues can have O-acetyl groups added to them. We demonstrate here that rat hepatocytes express 9-O-acetylated Sias mainly at the plasma membranes of both apical (bile canalicular) and basolateral (sinusoidal) domains. Golgi fractions also contain 9-O-acetylated Sias on similar N-linked glycoproteins, indicating that O-acetylation may take place in the Golgi. We show here that CMP-Sia-FITC (with a fluorescein group attached to the Sia) is taken up by isolated intact Golgi compartments. In these preparations, Sia-FITC is transferred to endogenous glycoprotein acceptors and can be immunochemically detected in situ. Addition of unlabeled UDP-Gal enhances Sia-FITC incorporation, indicating a substantial overlap of beta-galactosyltransferase and sialyltransferase machineries. Moreover, the same glycoproteins that incorporate Sia-FITC also accept [3H]galactose from the donor UDP-[3H]Gal. In contrast, we demonstrate with three different approaches (double-labeling, immunoelectron microscopy, and addition of a diffusible exogenous acceptor) that sialyltransferase and O-acetyltransferase machineries are much more separated from one another. Thus, 9-O-acetylation occurs after the last point of Sia addition in the trans-Golgi network. Indeed, we show that 9-O-acetylated sialoglycoproteins are preferentially segregated into a subset of vesicular carriers that concentrate membrane-bound, but not secretory, proteins.

Acetylation↗
PubMed1996

Subsets of sialylated, sulfated mucins of diverse origins are recognized by L-selectin. Lack of evidence for unique oligosaccharide sequences mediating binding.

Previous studies have shown that the mucin-type polypeptides GlyCAM-1, CD34, and MAdCAM-1 can function as ligands for L-selectin only when they are synthesized by the specialized high-endothelial venules (HEV) of lymph modes. Since sialylation, sulfation, and possibly fucosylation are required for generating recognition, we reasoned that other mucins known to have such components might also bind L-selectin. We show here that soluble mucins secreted by human colon carcinoma cells, as well as those derived from human bronchial mucus can bind to human L-selectin in a calcium-dependent manner. As with Gly-CAM-1 synthesized by lymph node HEV, alpha 2-3 linked sialic acids and sulfation seem to play a critical role in generating this L-selectin binding. In each case, only a subset of the mucin molecules is recognized by L-selectin. Binding is not destroyed by boiling, suggesting that recognition may be based primarily upon carbohydrate structures. Despite this, O-linked oligosaccharide chains released from these ligands by beta-elimination do not show any detectable binding to L-selectin. Following protease treatment of the ligands, binding persists in a subset of the resulting fragments, indicating that specific recognition is determined by certain regions of the original mucins. However, O-linked oligosaccharides released from the subset of non-binding mucin fragments do not show very different size and charge profiles compared to those that do bind. Furthermore, studies with polylactosamine-degrading endoglycosidases suggest that the core structures involved in generating binding can vary among the different ligands. Taken together, these data indicate that a single unique oligosaccharide structure may not be responsible for high-affinity binding. Rather, diverse mucins with sialylated, sulfated, fucosylated lactosamine-type O-linked oligosaccharides can generate high-affinity L-selectin ligands, but only when they present these chains in unique spacing and/or clustered combinations, presumably dictated by the polypeptide backbone.

Amino Sugars↗
PubMed1995

Unusual anionic N-linked oligosaccharides from bovine lung.

We previously described a diverse family of sulfated anionic N-linked oligosaccharides released by peptide: N-glycosidase F (PNGaseF) from calf pulmonary artery endothelial (CPAE) cells (Roux, L., Holoyda, S., Sundblad, G., Freeze, H.H., and Varki, A. (1988) J. Biol. Chem. 263, 8879-8889). Since a major fraction of the intact lung consists of endothelial cells, we reasoned that bovine lung might be a rich source of similar molecules. Total N-linked oligosaccharides from bovine lung acetone powder were released by PNGaseF, labeled by [3H]NaBH4 reduction, and the anionic fractions were studied with a variety of techniques. The sugar chains with lesser negative charge (designated Class I) share several properties of conventional multiantennary complex-type chains. However, unlike the case with CPAE cells, sialic acids account only for a minority of the anionic properties and only a small proportion carry sulfate esters. A variety of different treatments indicate that most of the unexplained negative charge is due to multiple carboxylic acid groups. Resistance to beta-glucuronidase and alpha-iduronidase suggests that these may be previously undescribed modifications of mammalian oligosaccharides. The most highly charged N-linked chains (designated Class II) are more similar in general structure to the corresponding ones from CPAE cells, although relatively more abundant. Their high charge is primarily due to chondroitin sulfate, heparin/heparan sulfate, or keratan sulfate glycosaminoglycan chains. Sequential digestion studies suggest that a significant proportion of these molecules have more than one type of glycosaminoglycan chain associated with them. Compositional analysis indicates the presence of xylose residues in Class II, but not Class I molecules. However, unlike the case with conventional glycosaminoglycans, these residues are not at the reducing terminus. Most previously reported structures of complex-type N-linked oligosaccharides are derived from the glycoproteins of blood cells, plasma, or the secretions of cultured mammalian cells. This library of N-linked oligosaccharides from an intact mammalian organ (lung) contains a high proportion of novel anionic sugar chains whose structures are different from conventional complex-type sialylated chains and only partially related to those from CPAE cells. Further exploration of the N-linked chains of intact mammalian tissues seems warranted.

Acetone↗
PubMed1995

Structure of the N-linked oligosaccharides of MHC class I molecules from cells deficient in the antigenic peptide transporter. Implications for the site of peptide association.

Class I molecules are N-linked glycoproteins encoded by the MHC. They carry cytosolic protein-derived peptides to the cell surface, displaying them to enable immune surveillance of cellular processes. Peptides are delivered to class I molecules by the transporter associated with Ag processing (TAP). Peptide association is known to occur before exposure of class I molecules to the medial Golgi-processing enzyme alpha-mannosidase II, but there is limited information regarding the location or timing of peptide binding within the earlier regions of the endoplasmic reticulum (ER)-Golgi pathway. A reported association of newly synthesized class I molecules with the ER chaperonin calnexin raises the possibility of persistence of the monoglycosylated N-linked oligosaccharide (NLO) Glc1Man8GlcNAc2, known to be recognized by this lectin. To explore these matters, we determined the structure of the NLOs on the subset of newly synthesized class I molecules awaiting the loading of peptide. We pulse-labeled murine MHC H-2Db class I molecules in RMA/S cells, which lack one of the TAP subunits, causing the great majority of the molecules to be retained for prolonged periods in an early secretory compartment, awaiting peptide binding. MHC molecules pulse-labeled with [3H]glucosamine were isolated, the NLOs specifically released and structurally analyzed by a variety of techniques. Within the chosen window of biosynthetic time, most Db molecules from parental RMA cells carried mature NLOs of the biantennary complex-type, with one to two sialic acid residues. In RMA/S cells, such chains were in the minority, the majority consisting of the precursor forms Man8GlcNAc2 and Man9GlcNAc2. No glucosylated forms were detected, nor were the later processing intermediates Man5-7GlcNAc2 or GlcNAc1Man4-5GlcNAc2. Thus, most Db molecules in TAP-deficient cells are retained in an early compartment of the secretory pathway, before the point of first access to the Golgi alpha-mannosidase I, which trims alpha 1-2 linked mannose residues, but beyond the point where the alpha 1-3-linked glucose residue is finally removed by the ER glucosidase II. Thus, structural analysis of NLOs on class I molecules within a defined biosynthetic window has established a biochemical measure of the timing of peptide association.

Animals↗
PubMed1995

Differential colon cancer cell adhesion to E-, P-, and L-selectin: role of mucin-type glycoproteins.

E-, P-, and L-selectin support the adhesion of leukocytes to the vessel wall through the recognition of specific carbohydrate ligands, which often contain sialylated, fucosylated lactosamines such as sialyl Lewis x [sLex; Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-]. E-selectin expressed by activated endothelium has been shown to support the adhesion of sLex-bearing colon cancer cells. In the present study, we examine the interactions of multiple colon cancer cell lines with all three selectins. Three colon cancer cell lines (LS 180, T84, and COLO 205) bound to recombinant purified E-, P-, and L-selectin. The colon cancer line COLO 320 bound to P- and L-selectin but not E-selectin; conversely, HT-29 cells bound E-selectin but not P- and L-selectin. Caco-2 showed little or no interaction with any of the three selectins. Treatment of the cells with O-sialoglycoprotease from Pasteurella haemolytica, an enzyme that selectively cleaves mucin-type O-linked glycoproteins, reduced binding to purified P- and L-selectin in all cases. In addition, recombinant soluble P- and L-selectin bound to affinity-purified mucins from all adherent tumor cell lines. Of the four tumor cell lines that interacted with E-selectin, O-glycoprotease treatment substantially diminished adhesion of LS 180 and T84, had little effect on COLO 205, and failed to inhibit the binding of HT-29. As predicted by these data, E-selectin showed substantial binding only to mucins purified from LS 180 and T84. These findings suggest that L- and P-selectin interact primarily with mucin-type ligands on colon cancers, whereas E-selectin can recognize both mucin and nonmucin ligands. Binding of the colon cancer lines to purified selectins correlates with their adhesion to activated endothelial cells (E-selectin-dependent), platelets (P-selectin-dependent), and neutrophils (L-selectin-dependent). These differential tumor cell-selectin interactions may influence metastatic spread and may also contribute to the observed variability in host response to tumor progression.

Animals↗
PubMed1995

I-type lectins.

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Amino Acid Sequence↗
PubMed1995

Endothelial heparan sulfate proteoglycans that bind to L-selectin have glucosamine residues with unsubstituted amino groups.

We earlier reported calcium-dependent, heparin-like L-selectin ligands in cultured bovine endothelial cells (Norgard-Sumnicht, K. E., Varki, N. M., and Varki, A. (1993) Science 261,480-483). Here we show that these are heparan sulfate proteoglycans (HSPGs) associated either with the cultured cells or secreted into the medium and extracellular matrix. Activation of the endothelial cells with bacterial lipopolysaccharide (LPS) does not markedly alter the amount or distribution of this material. A major portion of the glycosaminoglycan (GAG) chains released from these HSPGs by alkaline beta-elimination rebinds to L-selectin in the presence of calcium, indicating that these saccharides alone can mediate the high affinity recognition. Heparin lyase digestions indicate that these GAG chains are enriched in heparan sulfate, not heparin sequences. Current understanding of the biosynthesis of heparan sulfate chains indicates that all glucosamine amino groups must be either N-acetylated or N-sulfated. However, nitrous acid deamination at pH 4.0 suggests the presence of some unsubstituted amino groups in these L-selectin-binding GAG chains from endothelial cell HSPGs. This is confirmed by chemical N-reacetylation and by reactivity with sulfo-N-hydroxysuccinimide-biotin. These unsubstituted amino groups are also found on HSPGs from human umbilical vein endothelial cells, but are not detected in those from Chinese hamster ovary cells. In both bovine and human endothelial cells, these novel groups are enriched for in the HS-GAG chains which bind to L-selectin. Despite this, studies with N-reacetylation and nitrous acid deamination do not show conclusive evidence for the direct involvement of the unsubstituted amino groups in L-selectin binding. This may be because the chemical reactions used to modify the amino groups do not go to completion. Alternatively, the unsubstituted amino groups may only be indirectly involved in generating binding, by dictating the biosynthesis of another critical group. Regardless, these studies shown that HSPGs from cultured endothelial cells which can bind to L-selectin are enriched with unsubstituted amino groups on their GAG chains. The possible biochemical mechanisms for generation of these novel groups are discussed.

Acetylation↗
PubMed1995

Characterization of sialyloligosaccharide binding by recombinant soluble and native cell-associated CD22. Evidence for a minimal structural recognition motif and the potential importance of multisite binding.

CD22, a B cell-specific receptor of the immunoglobulin superfamily, has been demonstrated to bind to oligosaccharides containing alpha 2-6-linked sialic acid (Sia) residues. Previously, we demonstrated that the minimal structure recognized by this lectin is the trisaccharide Sia alpha 2-6Gal beta 1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell, L., and Varki, A. (1994) J. Biol. Chem. 269, 10628-10636). Here we utilize a soluble immunoglobulin fusion construct (CD22Rg) to determine directly by equilibrium dialysis the stoichiometry (2:1) and dissociation constant (32 microM) for Neu5Ac alpha 2-6Gal beta 1-4Glc binding. Inhibition assays performed with over 30 different natural and synthetic sialylated and/or sulfated compounds are utilized to define in greater detail specific structural features involved in oligosaccharide-protein binding. Specifically, the critical features required for binding include the exocyclic hydroxylated side chain of the Sia residue and the alpha 2-6 linkage position to the underlying Gal unit. Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter residue have limited effect on the binding. The nature of the residue to which the Gal is attached may affect binding. Bi(alpha 2-6)-sialylated biantennary oligosaccharides are capable of simultaneously interacting with both lectin sites present on the dimeric CD22Rg fusion construct, giving a marked improvement in binding over monosialylated compounds. Furthermore, data are presented indicating that full-length native CD22, expressed on the surface of Chinese hamster ovary cells, is structurally and functionally a multimeric protein, demonstrating a higher apparent affinity for multiply sialylated compounds over monosialylated compounds. These observations provide a mechanism for strong CD22-dependent cell adhesion despite the relatively low Kd for protein-sugar binding.

Animals↗
PubMed1995

CD22-mediated cell adhesion to cytokine-activated human endothelial cells. Positive and negative regulation by alpha 2-6-sialylation of cellular glycoproteins.

We previously showed that cultured human umbilical vein endothelial cells (HEC) exposed to the inflammatory cytokines tumor necrosis factor-alpha or interleukin-1 display increased activity of beta-galactoside alpha 2,6-sialyltransferase. This is associated with enhanced expression of ligands for the B cell receptor CD22 beta, which recognizes alpha 2-6-linked sialic acids (Hanasaki, K., Varki, A., Stamenkovic, I., and Bevilacqua, M. P. (1994) J. Biol. Chem. 269, 10637-10643). Here we report that increased expression of CD22 ligands is a feature of dermal microvascular endothelial cells as well, and is also observed in response to the cytokine interleukin-4. Tumor necrosis factor-alpha stimulation of HEC causes no change in the profile of endothelial glycoproteins recognized by CD22, but doubles the proportion of total cellular N-linked oligosaccharides capable of binding tightly to CD22. This modest change is sufficient to cause a marked increase in alpha 2-6-linked sialic acid-dependent binding of Chinese hamster ovary (CHO) cells expressing recombinant human CD22. In contrast, B lymphoma cell lines expressing higher levels of cell surface CD22 do not show such sialic acid-dependent binding to activated HEC. Since B lymphoma cells themselves also express high levels of alpha 2-6-linked sialic acids, their CD22 molecules might be rendered nonfunctional by endogenous ligands. In support of this, the lectin function of CD22 can be directly detected on transfected CHO cells, but not on B lymphoma cells. Furthermore, coexpression of beta-galactoside alpha 2,6-sialyltransferase with CD22 in the CHO cells abrogates sialic acid-dependent binding to cytokine-activated HEC. However, such co-transfected cells can bind to B lymphoma cells in a manner apparently less dependent upon alpha 2-6-linked sialic acid, suggesting CD22-mediated interactions that may not be directly dependent on its lectin function. Thus, CD22-mediated interactions between B cells and activated vascular endothelium may be positively regulated by induction of alpha 2-6-linked sialic acid-bearing endothelial cell ligands, but negatively regulated by such ligands on the B cells expressing CD22. Since expression of both CD22 and beta-galactoside alpha 2,6-sialyltransferase are regulated during B cell ontogeny, these findings could be of importance in B cell function and/or trafficking.

Animals↗
PubMed1995

Binding of human plasma sialoglycoproteins by the B cell-specific lectin CD22. Selective recognition of immunoglobulin M and haptoglobin.

CD22 is a cell-surface receptor of resting mature B cells that recognizes sialic acid (Sia) in the natural structure Sia alpha 2-6Gal beta 1-4GlcNAc (Powell, L. D., Jain, R. K., Matta, K. L., Sabesan, S., and Varki, A. (1995) J. Biol. Chem. 270, 7523-7532). Human umbilical vein endothelial cells (HEC) treated with inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) display increases in cell-surface CD22 ligands, caused by increased expression of the enzyme beta-galactoside alpha 2,6-sialyltransferase (Hanasaki, K., Varki, A., Stamenkovic, I., and Bevilacqua, M. P. (1994) J. Biol. Chem. 269, 10637-10643; Hanasaki, K., Varki, A., and Powell, L. D. (1995) J. Biol Chem. 270, 7533-7542). Thus, CD22 could direct potential interactions between mature B cells and endothelial cells during inflammatory states. However, this would have to occur in the presence of blood plasma, which contains many sialoglycoproteins known to carry alpha 2-6-linked sialic acids. We show here that human plasma can indeed inhibit Sia-dependent binding of a recombinant soluble chimeric form of human CD22 (CD22Rg) to TNF-alpha activated HEC. Affinity adsorption of individual human plasma samples with immobilized CD22Rg showed that, of the numerous alpha 2-6-sialic acid containing glycoproteins in plasma, only three polypeptides with apparent molecular mass (under reducing conditions) of 74, 44, and 25 kDa bound, and were specifically eluted with alpha 2-6-sialyllactose. NH2-terminal amino acid sequencing of these high affinity CD22 ligands revealed that they are subunits of immunoglobulin M (IgM) and haptoglobin. Purified human IgM from pooled human plasma can be quantitatively bound by CD22Rg, and binding is blocked by alpha 2-6-sialyllactose, but not by alpha 2-3-sialyllactose. Pretreatment by sialidase or by mild periodate oxidation of sialic acid side chains abolishes these interactions. IgM at physiological concentrations also inhibits CD22Rg binding to TNF-alpha-activated HEC in a manner dependent not only upon its sialylation but also requiring its intact multimeric structure. These data show that CD22 is capable of highly selective recognition of certain multimeric plasma sialoglycoproteins that carry alpha 2-6-linked sialic acids. Notably, the two proteins that are selectively recognized are known to be involved in immune and inflammatory responses. Haptoglobin synthesis by the liver is markedly increased during the "acute phase response" to systemic inflammation, while IgM is the major product resulting from activation of resting CD22-positive B cells.

Antigens, CD↗
PubMed1995

Expression of de-N-acetyl-gangliosides in human melanoma cells is induced by genistein or nocodazole.

Neuraminic acid is the core structure of most known sialic acids. In natural systems, the amino group at the 5 position of neuraminic acid residues is usually assumed to be acylated. Previously, synthetic de-N-acetyl-gangliosides (with free amino groups at the 5 position of neuraminic acids) have been shown to modulate cellular proliferation and tyrosine phosphokinase reactions. While indirect evidence has suggested that traces of these molecules exist naturally in certain tumor cells, further exploration has been hampered by the lack of a system showing consistent expression at an easily detectable level. Using synthetic compounds as antigens, we have developed highly specific monoclonal antibodies against de-N-acetyl-GM3 and de-N-acetyl-GD3 that require both the free amino group and the exocyclic side chain of sialic acids for recognition. Cultured human melanoma cells showed low but variably detectable levels of reactivity with these antibodies. The ability of various biologically active molecules to stimulate this reactivity was explored. Of many compounds tested, only the tyrosine kinase inhibitor genistein induced reactivity in a dose-dependent manner. Antibody reactivity with ganglioside extracts from genistein-treated cells was abolished by chemical re-N-acetylation and/or truncation of sialic acid side chains by mild periodate oxidation. High performance thin layer chromatography immuno-overlay analysis confirmed the presence of the novel compound de-N-acetyl-GD3 in these extracts. Several other tyrosine kinase inhibitors tested did not give the same increase in de-N-acetyl-ganglioside expression. However, the microtubule inhibitor nocodazole caused a similar accumulation of these molecules, particularly in non-adherent cells expected to be arrested at metaphase. Thus, genistein may induce de-N-acetyl-ganglioside expression by virtue of its known ability to arrest cells in the G2M phase, rather than as a general consequence of tyrosine kinase inhibition. These studies also provide a system in which to analyze the enzymatic basis of de-N-acetyl-ganglioside expression and their potential roles as growth regulating molecules.

Antibodies, Monoclonal↗
PubMed1995

Co-localization of hydrolytic enzymes with widely disparate pH optima: implications for the regulation of lysosomal pH.

Lysosomes are traditionally defined by their acidic interior, their content of degradative 'acid hydrolases', and the presence of distinctive membrane proteins. Terminal degradation of the N-linked oligosaccharides of glycoproteins takes place in lysosomes, and involves several hydrolases, many of which are known to have acidic pH optima. However, a sialic acid-specific 9-O-acetyl-esterase and a glycosyl-N-asparaginase, which degrade the outer- and inner-most linkages of N-linked oligosaccharides, respectively, both have pH optima in the neutral to alkaline range. By immunoelectron microscopy, these enzymes co-localize in lysosomes with several conventional acid hydrolases and with lysosomal membrane glycoproteins. Factors modifying the pH/activity profiles of these enzymes could not be found in lysosomal extracts. Thus, the function of the enzymes with neutral pH optima must depend either upon their minimal residual activity at acidic pH, or upon the possibility that lysosomes are not always strongly acidic. Indeed, when lysosomes are marked in living cells by uptake of fluorescently labeled mannose 6-phosphorylated proteins, the labeled organelles do not all rapidly accumulate Acridine Orange, a vital stain that is specific for acidic compartments. One plausible explanation is that lysosomal pH fluctuates, allowing hydrolytic enzymes with a wide range of pH optima to efficiently degrade macromolecules.

Acetylesterase↗
PubMed1994

Intramolecular self-cleavage of polysialic acid.

Polysialic acid (PSA) is an unusual homopolymer of sialic acid (Sia) found on a limited number of animal glycoproteins and in the capsules of certain pathogenic bacteria. The biological properties of PSA are known to vary markedly with the length of the polymer. We confirm here that while the primary linkage unit of PSA (Sia alpha 2-8Sia) is more stable than commoner Sia linkages, PSA with > 3 Sia units is substantially more labile. A "limit digest" of PSA yields fragments of degree of polymerization (DP) = 2 and 3 and little monomeric Sia. In keeping with this, the fragmentation of PSA of DP 4 is non-random, with the internal glycosidic bond being more labile than those at the two ends. The accelerated breakdown of PSA involves an intramolecular mechanism that is not explained by lactone formation, cation effects, or specific secondary structural features. However, it is dependent upon the intactness of internal carboxyl groups, which have an anomalously high pKa. Thus, the instability of PSA appears to result from intramolecular self-cleavage of the glycosidic bonds of internal Sia units, in which the adjacent carboxyl group with a high pKa acts as a proton donor for general acid catalysis. This lability of PSA is seen under mildly acidic conditions that can be encountered in various physiological and pathological situations and thus has potential implications for neuronal adhesion, embryogenesis, and bacterial pathogenicity.

Carbohydrate Sequence↗
PubMed1994

Selectin ligands.

The selectins initiate many critical interactions among blood cells. The volume of information and diversity of opinions on the nature of the biologically relevant ligands for selectins is remarkable. This review analyzes the matter and suggests the hypothesis that at least some of the specificity may involve recognition of "clustered saccharide patches."

Animals↗