Lennart Rodén

Structure and Metabolism of Connective Tissue Proteoglycans

With the possible exception of hyaluronic acid, the connective tissue polysaccharides are all synthesized by their parent cells as components of proteoglycans. In these substances, a number of polysaccharide chains are covalently linked to a protein core; e.g., in the proteoglycan of bovine nasal cartilage, which is the prototype of molecules of this kind, close to 100 chondroitin sulfate chains, with a molecular weight of approximately 20,000, and slightly fewer keratan sulfate chains are linked to a core protein (mol. wt. 200,000) which constitutes 7–8% of the entire molecule. In many respects, the proteoglycans are similar to other protein-bound complex carbohydrates, and the conspicuous polysaccharide component per se does not distinguish the proteoglycans from the class of glycoproteins; e.g., there are members of the glycoprotein class, such as the blood group substances, which have a high relative content of carbohydrate consisting of a substantial number of monosaccharide units. Rather, the segregation of the proteoglycans into a separate category is based on a few specific characteristics: (1) each polysaccharide consists of repeating disaccharide units in which a hexosamine, d-glucosamine, or d-galactosamine is always present; (2) all connective tissue polysaccharides except keratan sulfate contain a uronic acid, either d-glucuronic acid or its 5-epimer, l-iduronic acid, or both; (3) ester sulfate groups are present in all members of the group except in hyaluronic acid; in addition, N-sulfate groups are found in heparin and heparan sulfate. Although certain other bipolymers are known to contain ester sulfate, e.g., some epithelial mucins (Horowitz, 1977), these compounds are clearly distinguishable from the connective tissue polysaccharides by the other criteria indicated above. It may also be mentioned that the d-glucuronic-acid-containing repeating disaccharide of chondroitin, N-acetylchondrosine, has recently been identified as a component of thyroglobulin (Spiro, 1977); however, since the disaccharide is present as a single unit, thyroglobulin may not be considered a proteoglycan.

Biosynthesis of heparin

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.

Link protein and a hyaluronic acid-binding region as components of aorta proteoglycan

Summary Proteoglycans from bovine aorta were extracted with 4 M guanidine HCl in the presence of protease inhibitors and fractionated by density gradient centrifugation in CsCl. The bottom fraction from a dissociative gradient contained proteoglycans with a size distribution similar to that of cartilage proteoglycans. Also present were antigens which reacted with antisera raised against the hyaluronic acid-binding region and chondroitin sulfate peptides from cartilage proteoglycans. By a combination of associative and dissociative density gradient centrifugations, a fraction was obtained which was precipitated by antiserum to link proteins of bovine nasal cartilage and had the same electrophoretic mobility as link protein 1.

Separation of acidic oligosaccharides by gel filtration

Abstract Gel filtration has been used for the separation of the acidic oligosaccharides obtained by testicular hyaluronidase treatment of hyaluronic acid and of chondroitin 4-sulfate. In each series, the four lower members have been isolated and characterized by anasalysis before and after reduction with sodium borohydride.

Location of xylosyltransferase in the cisternae of the rough endoplasmic reticulum of embryonic cartilage cells.

Purified antibodies were prepared to UDP-D-xylose: core protein xylosyltransferase, the enzyme which initiates the formation of chondroitin sulfate chains in the course of proteoglycan biosynthesis in cartilage. The purified antibodies were conjugated to ferritin with a two-step glutaraldehyde procedure, and conjugates were then used to locate xylosyltransferase in fragments of embryonic cartilage cells. The results indicated that the enzyme is located within the cisternae of the rough endoplasmic reticulum. The distribution of the enzyme was similar to that of prolyl hydroxylase in the same cell fragments, suggesting that procollagen synthesis and initiation of chondroitin sulfate chains occur in the same regions of the rough endoplasmic reticulum.

Biosynthesis of chondroitin sulfate: Interaction between xylosyltransferase and galactosyltransferase

Abstract An affinity matrix consisting of the core protein of cartilage proteoglycan coupled to Sepharose was used to study the interaction between the glycosyltransferases which catalyze the first two reactions in the biosynthesis of chondroitin sulfate. Xylosyltransferase, for which the core protein is a substrate, is quantitatively adsorbed to the matrix. In contrast, UDP-galactose:xylose galactosyltransferase is not significantly adsorbed, but does bind to matrix which has been previously equilibrated with xylosyltransferase. By virtue of this enzyme-enzyme interaction, a 7-fold purification of galactosyltransferase can be obtained.

Heparin — an Introduction

Seventy-five years after its discovery (1,2), heparin remains an important tool in medicine. Among its established uses are the prevention of postoperative thrombosis, the treatment of acute venous thrombosis, and the prevention of clot formation in the heart-lung machine (3). Little was known about the structure of heparin when the first clinical trials began in the mid-1930s, and it is only during the 1980s that the detailed structural basis of heparin action has been elucidated through the characterization of a specific, antithrombin-binding pentasaccharide segment in the polysaccharide molecule. In the following, we shall retrace some of the steps that have led to this goal.

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.

Proteoglycans and Hypertension I.A Biochemical and Ultrastructural Study of Aorta Glycosaminoglycans in Spontaneously Hypertensive Rats

The extracellular matrix of blood vessel walls contains elastin, collagen, and proteoglycans, all of which can affect vascular resistance and, hence, blood pressure by virtue of their biomechanical properties. In the present study, we have begun to explore the possibility that proteoglycans may play a role in the pathophysiology of hypertension by analyzing, qualitatively and quantitatively, the polysaccharide components of proteoglycans from aorta of two normotensive rat strains, Wistar Kyoto (WKY) and Wistar rats, and from spontaneously hypertensive (SH) rats of the Okamoto strain. The total concentration of aorta glycosaminoglycans in the SH rat was 33% higher than in the WKY rat, due to a 164% increase in chondroitin 4- and 6-sulfate. The content of dermatan sulfate (DS), hyaluronic acid (HA), and heparan sulfate (HS) was similar in the two strains. The 4-wk-old SH rat also had an increase in chondroitin sulfate (CS) compared to the 4-wk-old WKY rat, without any change in DS, HA, or HS. The Wistar rat had approximately the same concentration of CS und DS in the aorta as the WKY rat, but HS und HA were reduced by 62 and 37%, respectively. The galactosaminoglycans (CS and DS) were heterogeneous on cellulose acetate electrophoresis and exhibited a different pattern for each of the three strains. Undersulfated CS accounted for 15% of the total CS in WKY aorta but was present in only trace amounts in the SH aorta; 2% of the CS from the Wistar aorta was undersulfated. In all three strains, DS was exclusively 4-sulfated, and the CS contained approximately equal amounts of 4- and 6-sulfated galactosamine residues. Ultrastructural studies demonstrated that the HS was localized in the subendothelial matrix and the pericellular region surrounding the medial smooth muscle cells. CS and DS were primarily associated with collagen in the media. In the SH rat aorta the subendothelial matrix was thicker, and there was a relative increase in the CS/DS in the smooth muscle cell pericellular matrix. We suggest that, if similar alterations in CS proteoglycans are present in the resistance vessels, these changes may contribute to the increased peripheral vascular resistance in the hypertensive animal.

Silk—A new substrate for UDP-d-xylose: Proteoglycan core protein β-d-xylosyltransferase☆

The formation of most connective tissue polysaccharides is initiated by transfer of D-xylose from UDP-D-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [14C]xylose transferred from UDP-D-[14C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 M NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the Vmax for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. Km values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [14C]xylose transfer to silk was alkali labile, and [14C]xylitol was formed when [14C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [14C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [14C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 M KOH; 100 degrees C; 8 h) and by acid hydrolysis which yielded [14C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had Vmax values 60-70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide. Ser-Gly-Ala-Gly-Ala-Gly; the latter had a Vmax value close to 20% of that of intact fibroin.