Milligram-scale preparation and purification of oligosaccharides of defined length possessing the structure of chondroitin from defructosylated capsular polysaccharide K4

Escherichia coli K4 bacterium synthesizes a nonsulfated capsule polysaccharide (K4) composed of a repeating disaccharide subunit of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid residues. The K4 polyanion is easily defructosylated under acid conditions with no fragmentation of the polymer to produce a polysaccharide having a repeated disaccharide unit of chondroitin consisting of D-glucuronic acid (beta1-->3) and N-acetyl-D-galactosamine (beta1-->4) (K4d). K4 and K4d were depolymerized by partial digestion with testicular hyaluronidase and separated into uniform-size oligosaccharides from 4-mers to 16-mers by preparative anion-exchange chromatography after removal of the hyaluronidase. The purity and size of each oligosaccharide was confirmed by using anion-exchange HPLC, HPSEC analysis, and FACE. Mg-scale K4d oligosaccharides were obtained from 50 mg K4d starting material. Under the conditions used to degrade the K4 polysaccharide by testicular hyaluronidase, fructose is slowly liberated forming the defructosylated K4. As a consequence, a mixture of uniform- size K4 and K4d oligosaccharide species, from approximately 4- to 20-mers, are generated and size-separated by anion-exchange chromatography. These pure, uniform-size, and large ranges of K4d oligosaccharides having the structure of a chondroitin, -->4)-GlcUA-beta(1-->3)GalNAc-beta(1-->, will be available for investigating important biological functions of this polymer.     

Preparation and application of biologically active fluorescent hyaluronan oligosaccharides

We report the production of biologically active hyaluronan (HA) oligosaccharides labeled with the fluorophore 2-aminobenzoic acid (2AA). Oligosaccharides from 4 to 40 residues in length were purified to homogeneity by ion exchange chromatography using a logarithmic gradient. Molecular weight and purity characterization of HA oligosaccharides is facilitated by 2AA derivatization because it enhances signals in MALDI-TOF MS and improves FACE (fluorophore-assisted carbohydrate electrophoresis) analysis by avoiding the inverted parabolic migration characteristic of 2-aminoacridone (AMAC)-labeled sugars. The small size and shape of the fluorophore maintains the biological activity of the derivatized oligosaccharides, as demonstrated by their ability to compete for polymeric HA binding to the G1-domain of human recombinant versican (VG1). An electrophoretic mobility shift assay was used to study VG1 binding to labeled HA 8-, 10-, 20-, 30-, and 40-mers, and although no stable VG1 binding was observed to labeled 8-mers, the equilibrium dissociation constant (100 nM) for VG1 with HA(10) was estimated from densitometry analysis of the free oligosaccharide. Interactions involving HA 20-, 30-, and 40-mers (proposed to be multivalent) could also be studied using this protocol. Oligosaccharides labeled with 2AA therefore show excellent potential as probes in fluorescence-based assays that investigate protein-carbohydrate interactions.     

Preparation and inhibitory activity on hyaluronidase of fully O-sulfated hyaluro-oligosaccharides

Hyaluronan was partially depolymerized on a large-scale quantity using bacterial hyaluronidase (E.C. 4.2.2.1) for preparation of chemically fully O-sulfated oligosaccharides. The hyaluro-oligosaccharide (HAoligo) mixture obtained by partial digestion was repeatedly applied to low pressure gel permeation chromatographic separation to purify the size-unified oligosaccharide ranged from 4- to 20-mer. The purity and size of each HAoligo was confirmed by using proton nuclear magnetic resonance ((1)H NMR) spectroscopy, capillary electrophoresis (CE) on normal polarity mode, and a newly established separation method by normal phase chromatography with Amide-80 column. The purified HAoligos ranged 4- to 20-mer were applied to chemically fully O-sulfation. Characterization of chemically fully O-sulfated HAoligos was performed by both chemical compositional analyses after hydrolysis and (1)H NMR spectroscopy. While the anti-factor IIa activity of 4- to 20-mer O-sulfated HAoligos was less than 3.1 units/mg, the inhibitory action for hyaluronidase (bovine testicular hyaluronidase (E.C.3.2.1.35)) of the oligosaccharides ranged 16- to 20-mer were corresponding to 79% of that shown by fully O-sulfated hyaluronan (MW 100 kDa) through both competitive and noncompetitive effects.     

Importance of hyaluronan length in a hyaladherin-based assay for hyaluronan

Specific hyaladherin-based assays have been set up to measure the concentration of hyaluronan in biological fluids. Hyaluronectin (HN; a hyaladherin extracted from ovine brain) binds to hyaluronan (HA) that must be 10 units (HA10) or more long. It was therefore of interest to determine whether HN would continue to bind to HA10 in full-length HA since conformational changes might mask potential binding sites. We used the enzyme-linked sorbent assay (ELSA) to assay HA and hyaluronan-derived oligosaccharides, with different standard HAs, and the results were compared to results obtained with the carbazole technique. Oligosaccharide length was calculated from the ratio glucuronic acid/reducing N-acetylglucosamine in fractions of hyaluronidase-digested macromolecular hyaluronan prepared by chromatography; the size of the HA12 oligosaccharide was confirmed by matrix-assisted laser desorption ionization mass spectrometry. During the digestion of macromolecular HA with hyaluronidase, the binding of HN to HA first increased and then decreased as shown using the ELSA. The concentration of HA fragments of HA60 and below was overestimated when intact macromolecular HA was used as the reference for the ELSA, while the concentration of HA100 and above was underestimated when HA10 was used as the reference. The binding of HN to HA20, HA40, and HA60 saccharides was consistent with binding to multiples of HA10 sites. In conclusion, the level of HN binding is determined by the conformation of HA, which may mask binding sites. Hence, calibration HA used in the ELSA must be adapted to the size of HA to assay.     

Microscale preparation of even- and odd-numbered N-acetylheparosan oligosaccharides

In order to prepare a series of N-acetylheparosan (NAH)-related oligosaccharides, bacterial NAH produced in Escherichia coli strain K5 was partially depolymerized with heparitinase I into a mixture of even-numbered NAH oligosaccharides, having an unsaturated uronic acid (DeltaUA) at the non-reducing end. A mixture of odd-numbered oligosaccharides was derived by removing this DeltaUA in the aforementioned mixture by a 'trimming' reaction using mercury(II) acetate. Each oligosaccharide mixture was subjected to gel-filtration chromatography to generate a series of size-uniform NAH oligosaccharides of satisfactory purity (assessed by analytical anion-exchange HPLC), and their structures were identified by MALDITOF-MS, ESIMS, and 1H NMR analysis. As a result, a microscale preparation of a series of both even- and odd-numbered NAH oligosaccharides was achieved for the first time. The developed procedure is simple and systematic, and thus, should be valuable for providing not only research tools for heparin/heparan sulfate-specific enzymes and their binding proteins, but also precursor substrates with medical applications.     

Hyaluronan oligosaccharides induce CD44 cleavage and promote cell migration in CD44-expressing tumor cells

CD44 is an adhesion molecule that serves as a cell surface receptor for several extracellular matrix components, including hyaluronan (HA). The proteolytic cleavage of CD44 from the cell surface plays a critical role in the migration of tumor cells. Although this cleavage can be induced by certain stimuli such as phorbol ester and anti-CD44 antibodies in vitro, the physiological inducer of CD44 cleavage in vivo is unknown. Here, we demonstrate that HA oligosaccharides of a specific size range induce CD44 cleavage from tumor cells. Fragmented HA containing 6-mers to 14-mers enhanced CD44 cleavage dose-dependently by interacting with CD44, whereas a large polymer HA failed to enhance CD44 cleavage, although it bound to CD44. Examination using uniformly sized HA oligosaccharides revealed that HAs smaller than 36 kDa significantly enhanced CD44 cleavage. In particular, the 6.9-kDa HA (36-mers) not only enhanced CD44 cleavage but also promoted tumor cell motility, which was completely inhibited by an anti-CD44 monoclonal antibody. These results raise the possibility that small HA oligosaccharides, which are known to occur in various tumor tissues, promote tumor invasion by enhancing the tumor cell motility that may be driven by CD44 cleavage.     

Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin.

The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.     

Heparin oligosaccharide sequence and size essential for inhibition of pulmonary artery smooth muscle cell proliferation

Heparin has a wide range of important biological activities including inhibition of pulmonary artery smooth muscle cell proliferation. To determine the minimum size of the heparin glycosaminoglycan chain essential for antiproliferative activity, porcine intestinal mucosal heparin was partially depolymerized with heparinase and fractionated to give oligosaccharides of different sizes. The structure of these oligosaccharides was fully characterized by 1D and 2D 1H NMR spectroscopy. These oligosaccharides were assayed for antiproliferative effects on cultured bovine pulmonary artery smooth muscle cells (PASMCs). The tetrasaccharide (4-mer) exhibited no heparin-like activity. Decasaccharides (10-mers) and dodecasaccharides (12-mers) displayed a reduced level of activity when compared to full-length heparin. Little effect on activity was observed in deca- and dodecasaccharides with one less 2-O-sulfo group. The 14-, 16-, and 18-mers showed comparable growth-inhibition effects on PAMSC as porcine intestinal mucosal heparin. These data suggest that a 14-mer is the minimum size of oligosaccharide that is essential for full heparin-like antiproliferative activity. Since the 14- to 18-mers have no 3-O-sulfo groups in their glucosamine residues, their full activity confirms that these 3-O-sulfonated glucosamine residues, which are required for heparin's anticoagulant activity, are not an essential requirement for antiproliferative activity.     

Novel products in hyaluronan digested by bovine testicular hyaluronidase

When the products of hyaluronan (HA) digested by bovine testicular hyaluronidase (BTH) were analyzed by high-performance liquid chromatography (HPLC), minor peaks were detected just before the main even-numbered oligosaccharide peaks. The amount of each minor peak was dependent on the reaction conditions for transglycosylation, rather than hydrolysis, by the BTH. Mainly based on HPLC and MS analysis, each minor peak was found to correspond to its oligosaccharide with one N-acetyl group removed from the reducing terminal N-acetylglucosamine. Enzymatic studies showed that the N-deacetylation activity was closely related to reaction temperature, pH, and the concentration of NaCl contained in the buffer, and glycosaminoglycan types and chain lengths of substrates. These findings strongly suggest that the N-deacetylation reaction in minor peaks was due to a novel enzyme contaminant in the BTH, N-deacetylase, that carries out N-deacetylation at the reducing terminal N-acetylglucosamine of oligosaccharides and is dependent on HA hydrolysis by BTH.     

Exploration of the action pattern of Streptomyces hyaluronate lyase using high-resolution capillary electrophoresis

Hyaluronic acid was treated exhaustively with a hyaluronate lyase (hyaluronidase, EC 4.2.2.1) from Streptomyces hyalurolyticus to obtain a tetrasaccharide and a hexasaccharide product in a molar ratio of 1 to 1.2. The tetrasaccharide product was fluorescently labeled at the reducing end by reductive amination with 7-amino 1,3-naphthalene disulfonic acid (AGA) and the structure of the conjugate was determined spectroscopically. Partial treatments of hyaluronic acid with hyaluronate lyase afforded complex mixtures of oligosaccharides that were similarly fluorescently labeled. These labeled oligosaccharide mixtures were analyzed using high-resolution capillary electrophoresis. The resulting electropherograms showed the content of each hyaluronic acid derived oligosaccharide, having a degree of polymerization (dp) from 4 to 50, throughout the enzymatic reaction. Computer simulation studies gave comparable kinetic profiles suggesting that hyaluronate lyase exhibits a random endolytic action pattern. Interestingly, oligosaccharides of certain size (dp) were under-represented in these oligosaccharide mixtures suggesting that linkages at spacings of 10 to 12 saccharide units are somewhat resistant to this enzyme. The cause of this resistance might be the result of secondary or higher order structural features present in the hyaluronic acid polymer.     

Human fibrinogen specifically binds hyaluronic acid

Fibrin and hyaluronic acid (HA) are macromolecules whose concentrations are elevated at the same time in the extracellular space of damaged tissues. We have investigated whether HA can bind to fibrinogen using solid phase and soluble assays. Purified human fibrinogen specifically bound to HA-Sepharose to a greater extent (greater than 5-fold) than did alpha 1-acid glycoprotein, DNaseI, ovalbumin, haptoglobin, or lysozyme. Fibrinogen did not bind to ethanolamine-Sepharose, a control chromatographic support. Treatment of HA-Sepharose containing bound 125I-fibrinogen with ovine testicular hyaluronidase released 44% of the 125I radioactivity, indicating that fibrinogen was specifically bound to HA. Moreover, 125I-fibrinogen bound to HA-Sepharose could be displaced by free HA but not by either of the monosaccharide components of this polymer, glucuronic acid, or N-acetylglucosamine. Chondroitin sulfate and polygalacturonic acid competed only weakly for bound 125I-fibrinogen. Bound 125I-fibrinogen was also not released by high concentrations of NaCl (up to 4 M), indicating that the interaction is not simply ionic. The apparent affinity of fibrinogen for HA covaried with the molecular weight of the HA. Small HA oligosaccharides (Mr = 3900) were only 50% as effective as larger HA (Mr = 8 X 10(5)) in eluting bound 125I-fibrinogen from HA-Sepharose. The optimal oligosaccharide size for displacement of bound 125I-fibrinogen was greater than or equal to 200 monosaccharides. Additionally, the amount of 125I-fibrinogen bound to HA-Sepharose was directly related to the size of the HA-amine linked to the affinity support. The affinity constant for fibrinogen binding to 125I-HA (approximately 150 monosaccharides) is estimated to be at least 2 X 10(7) M-1. These results demonstrate for the first time a specific, reversible binding between HA and fibrinogen.     

Lectin affinity high-performance liquid chromatography: interactions of N-glycanase-released oligosaccharides with leukoagglutinating phytohemagglutinin, concanavalin A, Datura stramonium agglutinin, and Vicia villosa agglutinin

We have developed a lectin affinity high-performance liquid chromatography technique for analysis of oligosaccharides using columns of silica-bound lectins. Purified leukoagglutinating phytohemagglutinin (L-PHA), concanavalin A (Con A), Datura stramonium agglutinin (DSA), and Vicia villosa agglutinin (VVA) were covalently coupled to periodate-oxidized diol-silica by reductive amination. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with the silica-bound lectins. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. The oligosaccharide specificities displayed by silica-bound L-PHA, Con A, and DSA were virtually identical to those established utilizing lectin-agarose conjugates. Analysis of oligosaccharides by lectin affinity HPLC allowed further definition of the specificity of VVA for N-glycanase-released, reduced oligosaccharides. Lectin affinity HPLC is rapid and convenient, providing an important structure-specific dimension to oligosaccharide analysis. This technique is particularly useful when utilized in conjunction with anion-exchange and ion-suppression amine adsorption HPLC methods, which fractionate on the basis of charge and size, respectively. In addition to their utility for oligosaccharide characterization, these affinity columns demonstrate the high degree of oligosaccharide specificity displayed by plant and animal lectins.     

Control of enzymatic degradation of hyaluronan by divalent cations.

Enzymatic degradation of hyaluronan (HA) by testicular hyaluronidase (HAase, hyaluronate 4-glucanohydrolase) requires inclusion of mono- or divalent cations in the reaction mixture. Most divalent cations activated HAase with equal potency; however, Cu2+ suppressed degradation, and Ca2+ showed a concentration-dependent regulation of size of the oligosaccharide products. Careful selection of HAase assay parameters is critical for discovery of novel HAase inhibitors and for preparation of controlled-size oligosaccharide fragments.     

Comparative structural study of N-linked oligosaccharides of urinary and recombinant erythropoietins

The structures of the N-linked oligosaccharides of the urinary erythropoietin (u-EPO) purified from urine of aplastic anemic patients were analyzed and compared with those for recombinant erythropoietin (r-EPO) prepared with baby hamster kidney (BHK) cells. Asparagine-linked neutral oligosaccharides were released from each EPO protein by N-oligosaccharide glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high-performance liquid chromatography (HPLC) on an ODS silica column. More than 8 and 13 kinds of oligosaccharide fractions for u-EPO and r-EPO (BHK), respectively, were completely separated by the one-step HPLC procedure. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amide-silica column. Furthermore, high-resolution proton nuclear magnetic resonance (1H NMR) spectroscopy and methylation analyses were carried out in the case of r-EPO (BHK).(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective hydrolysis of chondroitin sulfates by hyaluronidase

Chondroitin 4-sulfate and chondroitin 6-sulfate were incubated with testicular hyaluronidase in the presence of excess beta-glucuronidase. The beta-glucuronidase caused rapid removal of the nonreducing terminal beta-D-glucuronosyl residues from the oligosaccharides formed by the action of the hyaluronidase, destroying the oligosaccharide acceptors required for the transglycosylation activity of hyaluronidase and releasing free D-glucuronic acid at a rate that was equal to the rate of the hyaluronidase-catalyzed hydrolysis. When hyaluronidase was assayed at 37 degrees C in the presence of 0.05 M NaCl, 0.05 M Na2SO4, and 0.1 M sodium acetate at pH 5, chondroitin 4-sulfate was hydrolyzed at 1.5 times the rate found for chondroitin 6-sulfate. When hyaluronidase was assayed at 45 degrees C in 0.06 M sodium acetate at pH 6, chondroitin 4-sulfate was hydrolyzed at 8 times the rate observed for chondroitin 6-sulfate. Under the pH5 conditions, the chondroitin 4-sulfate was converted to a mixture of tri- and pentasaccharides, while the chondroitin 6-sulfate was converted primarily to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide. Under the pH 6 conditions, the chondroitin 4-sulfate was converted to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide, but the products from chondroitin 6-sulfate were a mixture of oligosaccharides ranging in degree of polymerization from 7 to 25 monosaccharides per oligosaccharide. End-group analyses of the products formed at pH 6 showed that both substrates were cleaved preferentially at the glycosidic bonds of the 4-sulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of a novel hyaluronidase from Streptococcus zooepidemicus in Escherichia coli and its application for the preparation of HA oligosaccharides

Hyaluronan (HA) oligosaccharides which can stimulate angiogenesis and suppress the growth of tumors have attracted more and more attention. In order to prepare pure and well-defined oligosaccharides from high-molecular-weight HA in a rapid and simple manner, an enzymatic degradation method was developed, which included degradation with a novel recombinant hyaluronan lyase (HA lyase, hyaluronidase, or HAase) and gel permeation chromatography. The HAase protein was expressed in Escherichia coli with the expression vector pBV220. The HAase was purified and refolded, and specific activity of the enzyme solution was 3800 U/mg. HA was degraded with HAase at the optimized conditions, yielding 46% and 31% of HA disaccharides and HA tetrasaccharides, respectively. These HA oligosaccharides were conveniently separated by consecutive column chromatography on Bio-gel P6 and were identified by HPLC–MS.     

Isoenzyme-specific differences in the degradation of hyaluronic acid by mammalian-type hyaluronidases

Bovine testicular hyaluronidase (BTH) has been used as a spreading factor for many years and was primarily characterized by its enzymatic activity. As recombinant human hyaluronidases are now available the bovine preparations can be replaced by the human enzymes. However, data on the pH-dependent activity of hyaluronidases reported in literature are inconsistent in part or even contradictory. Detection of the pH-dependent activity of PH-20 type hyaluronidases, i.e. recombinant human PH-20 (rhPH-20) and BTH, showed a shift of the pH optimum from acidic pH values in a colorimetric activity assay to higher pH values in a turbidimetric activity assay. Contrarily, recombinant human Hyal-1 (rhHyal-1) and bee venom hyaluronidase (BVH) exhibited nearly identical pH profiles in both commonly used types of activity assays. Analysis of the hyaluronic acid (HA) degradation products by capillary zone electrophoresis showed that hyaluronan was catabolized by rhHyal-1 continuously into HA oligosaccharides. BTH and, to a less extent, rhPH-20 exhibited a different mode of action: at acidic pH (pH 4.5) HA was degraded as described for rhHyal-1, while at elevated pH (pH 5.5) small oligosaccharides were produced in addition to HA fragments of medium molecular weight, thus explaining the pH-dependent discrepancies in the activity assays. Our results suggest a sub-classification of mammalian-type hyaluronidases into a PH-20/BTH and a Hyal-1/BVH subtype. As the biological effects of HA fragments are reported to depend on the size of the molecules it can be speculated that different pH values at the site of hyaluronan degradation may result in different biological responses.     

Structural study of asparagine-linked oligosaccharide moiety of taste-modifying protein, miraculin

The structures of the N-linked oligosaccharides of miraculin, which is a taste modifying glycoprotein isolated from miracle fruits, berries of Richadella dulcifica, are reported. Asparagine-linked oligosaccharides were released from the protein by glycopeptidase (almond) digestion. The reducing ends of the oligosaccharide chains thus obtained were aminated with a fluorescent reagent, 2-aminopyridine, and the mixture of pyridylamino derivatives of the oligosaccharides was separated by high performance liquid chromatography (HPLC) on an ODS-silica column. More than five kinds of oligosaccharide fractions were separated by the one chromatographic run. The structure of each oligosaccharide thus isolated was analyzed by a combination of sequential exoglycosidase digestion and another kind of HPLC with an amidesilica column. Furthermore, high resolution proton nuclear magnetic resonance (1H NMR) measurements were carried out. It was found that 1) five oligosaccharides obtained are a series of compounds with xylose-containing common structural core, Xyl beta 1----2 (Man alpha 1----6) Man beta 1----4-GlcNAc beta 1----4 (Fuca1----3)GlcNAc, 2) a variety of oligosaccharide structures are significant for two glycosylation sites, Asn-42 and Asn-186, and 3) two new oligosaccharides, B and D, with unusual structures containing monoantennary complex-type were characterized. (formula; see text)     

Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen.

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.     

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. We identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). We also developed 125I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125I-wheat germ agglutinin. 125I-labeled Ricinus communis agglutinin I and 125I-peanut agglutinin blotting of the desialylated proteins revealed few if any conventional O-linked oligosaccharides, suggesting that the sialyl residues represent termini of N-linked complex-type oligosaccharides. Depending upon the protein, we estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains. The labeling of these three proteins by 125I-concanavalin A was sensitive to treatment with endoglycosidase H, and each exhibited a quantitative reduction in Mr after the treatment, as assessed independently by 125I-wheat germ agglutinin blotting. At this level of analysis, we were unable to discern differences in the types of oligosaccharides present on these seven glycoproteins that correlate with their patterns of expression within the plasma membrane domains of this polarized epithelial cell.