Johan Riesenfeld

N-[3H]acetyl-labeling, a convenient method for radiolabeling of glycosaminoglycans

Abstract A method for the introduction of N -[ 3 H]acetyl groups into glycosaminoglycans is described. The procedure is based on [ 3 H]acetylation of N -unsubstituted hexosamine residues by treating the polysaccharides with [ 3 H]acetic anhydride. Preparations of heparin and heparin sulfate were found to contain significant numbers of N -unsubstituted hexosamine residues, as isolated. In contrast, such units could not be detected in chondroitin sulfate, dermatan sulfate, or hyaluronic acid. These polysaccharides were therefore subjected to partial N -deacetylation by reaction with hydrazine in the presence of hydrazine sulfate. After treatment with [ 3 H]acetic anhydride, the specific activities of the resulting labeled polysaccharide preparations ranged between 0.1 × 10 6 and 0.6 × 10 6 cpm 3 H/μg of uronic acid. The 3 H-labeled polysaccharide preparations did not differ significantly from the corresponding unlabeled starting materials with regard to polyanion properties (chromatography on DEAE-cellulose) or polymer chain size (gel chromatography). Further, the radiolabeled polysaccharide derivatives were susceptible to specific enzymatic degradation (chondroitinase ABC and mammalian heparitinase) and retained their ability to interact specifically with certain proteins—for example, [ 3 H]heparin with antithrombin [ 3 H]hyaluronic acid oligosaccharides with chondroitin sulfate proteoglycan (Cleland, R. L. Biochem. Biophys. Res. Commun. 87 , 1140–1145 (1979)). These findings indicate that the labeling procedures did not induce any major structural derangement of the polysaccharide molecules. The method developed should be useful in providing labeled glycosaminoglycans for metabolic and enzymatic experiments as well as for studies on the interaction between glycosaminoglycans and other biological macromolecules.

The molecular size of the antithrombin-binding sequence in heparin

The blood anticoagulant activity of heparin depends on its ability to bind, and thereby activate, antithrombin, a plasma protein that inhibits the proteinases of the so-called coagulation cascade [ 11. Only a fraction (-1/3rd) of the molecules in heparin preparations binds with high affinity to antithrombin and this fraction accounts for most of the anticoagulant activity of the unfractionated material [2-41. Attempts to define the structural basis for the interaction between heparin and antithrombin led to partially conflicting results. While it was claimed that the heparin structure required for binding to antithrombin is contained within a tetrasaccharide sequence [5], we proposed a more extended binding region [6]. Our conclusion was based on the isolation of oligosaccharides with high affinity for antithrombin, following partial depolymerization of heparin with bacterial heparinase [7] or with nitrous acid [6]. The oligosaccharides were tentatively identified as dodecaor tetradecasaccharides. However, no attempt was made to define the extent of the actual binding sequence by selecting for the smallest possible oligosaccharide yet capable of binding with high affinity to antithrombin. Here, such a component has been isolated and identified as an octasaccharide. The location in the octasaccharide molecule of a tetrasaccharide structure (IdUA+GlcNac+GlcUA+GlcNSOi), implicated in the antithrombin-binding sequence [5,6], has been determined.

Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides☆

A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505-1510); this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH approximately 1.5 (J. E. Shively, and H.E. Conrad, 1976, Biochemistry 15, 3932-3942) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH approximately 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH approximately 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5-8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.

Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate.

A new substrate for the deacetylase which catalyzes the removal of the N-acetyl groups from N-acetylheparosan in the course of heparin biosynthesis has been prepared. The capsular polysaccharide from Escherichia coli 010:K5:H4, which is structurally identical to N-acetylheparosan, was partially N-deacetylated by hydrazinolysis and was then radioactively labeled by N-acetylation with [3H]acetic anhydride. Upon incubation of the labeled polysaccharide with microsomes from the Furth mastocytoma, [3H]acetyl groups were released, demonstrating that the bacterial polysaccharide was a substrate for the N-deacetylase. Reaction conditions were established which permitted the quantitative assay of N-deacetylase activity; a Km of 74 mg polysaccharide/liter was determined, which corresponds to 2.1 X 10(-4) M, expressed as concentration of uronic acid; Vmax was 3.4 nmol/mg protein/liter. In confirmation of previous results, it was observed (a) that the reaction was stimulated by 3-phosphoadenylylsulfate (up to a maximum of 45% at a concentration of 0.5 mM), suggesting that N-sulfation occurred which facilitated continued action of the N-deacetylase, and (b) that NaCl and KCl inhibited the enzyme, with 50% reduction of activity at a concentration of 25 mM. In the course of this work, a simple, single-vial assay procedure was used. Released [3H]acetate was extracted from the acidified reaction mixture with a toluene- or xylene-based scintillation fluid containing 10% isoamyl alcohol and measured directly by scintillation spectrometry.

Biosynthesis of Heparin Effect of Detergent on the Microsomal Polymerization and Polymer Modification Processes

The formation of labeled heparin-precursor polysaccharide (N-acetylheparosan) from the nucleotide sugars, UDP-[14C]glucuronic acid and UDP-N-acetylglucosamine, in a mouse mastocytoma microsomal fraction was abolished by the addition of 1% Triton X-100. In contrast, the detergent-treated microsomal preparation retained the ability to convert such preformed polysaccharide into sulfated products during incubation with 3′-phosphoadenylylsulfate (PAPS). However, as shown by ion-exchange chromatography of these products, the detegent treatment changed the kinetics of sulfation from the rapid, repetivive process characteristic of the unperturbed system to a slow, progressive sulfation, which involved all polysarccharide molecules simultaneously and yielded, ultimately, a more highly sulfated product. The detergent effect was attributed to solubilization of sulfotransferases from the microsomal membranes, along with other polymer-modifying enzymes and the polysaccharide substrate. The resulting product showed an apparently random distribution ofN-acetyl andN-sulfate groups, instead of the predominantly block-wise arrangement achieved through membrane-associated biosynthesis.O-Sulfation occurred mainly at C2 of the iduronic acid units in the membrane-bound polysaccharide but at C6 of the glucosamine residues in the presence of detergent.A capsular polysaccharide fromEscherichia coli K5, previously found to have a structure identical to that of the nonsulfated heparin-precursor polysaccharide, was sulfated in the solubilized system in a fashion similar to that of the endogenous substrate, but was not accessible to the membrane-bound enzymes.These findings suggest that the regulation of the polymer-modification process, and hence the structure of the final polysaccharide product, depends heavily on the organization of the enzymes and their proteoglycan substrate in the endoplasmic membranes of the cell.