Alfred Linker

The heparitin sulfates (heparan sulfates).

Heparitin sulfate has been isolated from several sources, namely: a commercial lung polysaccharide preparation, beef lung, beef aorta, human amyloid liver, human intestine, and urine of a patient with mucopolysaccharidosis. The polysaccharides isolated were extensively purified, fractionated, and characterized. The data obtained show that heparitin sulfate is not a single compound but constitutes a family of related polymers which differ in sulfate content and in the arrangement of charged groups. These compounds are readily distinguished from most other glycosamino-glycuronans (mucopolysaccharides) by composition and optical rotation. They can be differentiated from heparin by their content of sulfate and N-acetyl groups, and D-glucuronic acid residues and by the ratio of the carbazole to orcinol uronic acid values. Due to the variations of charge distribution and molecular size, the heparitin sulfates are found to a considerable extent in admixture with other acidic polysaccharides during isolation and fractionation procedures. The particular type of heparitin sulfate obtained varies considerably with the tissue of origin. The lung-derived material was found to contain the largest range of subfractions. The heparitins isolated from aorta and amyloid liver were fairly homogenous in themselves, but differed from each other.

The structure of a polysaccharide from infectious strains of Burkholderia cepacia.

The structure of an acidic exopolysaccharide (EPS) from eight strains of Burkholderia cepacia has been investigated by methylation and sugar analysis, periodate oxidation-Smith degradation, and partial acid-hydrolysis. An enzyme preparation obtained from the same organisms producing the EPS was also used to depolymerize the polysaccharide. Detailed NMR studies of the chemical and enzymatic degradation products showed that this EPS consists of a highly branched heptasaccharide-repeating unit with the following structure: [abstract: see text]. About three O-acetyl groups per repeating unit are present at undetermined positions.

The disaccharide repeating-units of heparan sulfate

Abstract Five disaccharides have been isolated after degradation of heparan sulfate by heparinase (heparin lyase) and heparitinase (heparan sulfate lyase) and are suggested to represent the repeating units of the polysaccharide. They all contain a 4,5-unsaturated uronic acid residue and are: (a) A trisulfated disaccharide that is apparently identical to a disaccharide repeating-unit of heparin; (b) a disulfated disaccharide that seems unique for heparan sulfate and contains 2-deoxy-2-sulfamidoglucose and uronic acid sulfate residues; (c) a nonsulfated disaccharide containing a 2-acetamido-2-deoxyglucose residue; (d) a monosulfated disaccharide containing a 2-acetamido-2-deoxyglucose sulfate residue; and (e) a monosulfated disaccharide containing a 2-deoxy-2-sulfamidoglucose residue. Yields of these disaccharides from different heparan sulfate fractions are discussed in relation to possible arrangements of these units in the intact polymer.

Structural studies of heparitin sulfates.

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain. The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.

Structural studies on heparin. Tetrasaccharides obtained by heparinase degradation

Three tetrasaccharides representing major structural sequences of heparin were isolated in good yield and characterized after degradation of heparin by purified flavobacterial heparinase. N-Desulfation was necessary to achieve good separation of these closely related compounds from each other. One of the tetrasaccharides was shown to be derived from the fully sulfated repeating segments; to contain L-iduronic acid and six sulfate groups, and have the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4)-(6-SO4). The second contained a D-glucuronic acid unit that was nonsulfated instead of the L-iduronic acid, and the third, obtained in a fairly low yield, contained five sulfate groups, three of which being located on the disaccharide at the nonreducing end, and having the structure delta 4,5- HexpA -(2-SO4)-(1----4)-alpha-D- GlcpN -(N-SO4)-(6-SO4)-(1- ---4)-alpha -L- IdopA -(2-SO4)-(1----4)-D- GlcN -(N-SO4). All tetrasaccharides had a sulfated, unsaturated uronic acid unit at the nonreducing end, confirming that the heparinase requires sulfated L-iduronic acid units for activity.

Anticoagulant activity of heparin: Isolation of antithrombin-binding sites

The.anticoagulant activity of heparin is due to its ability to specifically bind to antithrombin and thus strikingly accelerate the rate of inactivation of a number of the enzymes involved in the coagulation mechanism [ 1,2] . It was recently demonstrated that heparin may be separated into distinct fractions, having high and low affinity, respectively, for antithrombin [3,4] ; the high-affinity fraction showed exceedingly high antigoagulant activity, whereas the low-affinity fraction was almost inactive. A preliminary structural characterization of the two heparin fractions failed to reveal any obvious dissimilarities [4]. In the present study heparin fragments capable of binding to antithrombin were isolated after digestion of heparin-antithrombin complexes with a bacterial heparinase. Fragments with high affinity for antithrombin were derived from high-affinity heparin only. They had a molecular weight of about 4-5 X 103. These findings suggest that binding of heparin to antithrombin may require a specific sequence of variously substituted sugar residues.

Hyaluronidase activity in leeches (Hirudinea)

The leech hyaluronoglucuronidase (hyaluronidase I) was identified in Erpobdellidae (Nephelopsis obscura and Erpobdella punctata) and Glossiphoniidae (Desserobdella picta) and historically described from Hirudinidae (Hirudo medicinalis). A second leech hyaluronidase (hyaluronidase II) which hydrolyzed only a few bonds to for hyaluronan oligosaccharides larger than 6500 Da, was found in Glossiphoniidae (Helobdella stagnalis, Glossiphonia complanata, Placobdella ornata, and Theromyzon sp.) and in Haemopidae (Haemopis marmorata). The distribution of the two hyaluronidases in leech occurred in both orders (Arhynchobdellida and Rhynchobdellida) and in macrophagous and haematophagous feeding types whereas the liquidosomatophagous leeches only had hyaluronidase II.

DISTINCTION AMONG FOUR FORMS OF HURLER'S SYNDROME.

Summary Analysis of the urinary AMPS from individuals with Hurlers syndrome indicates there are 4 forms that can be distinguished by their excretion pattern. The classical autosomal recessive form was shown to excrete significantly more chondroitin sulfate B in proportion to heparitin sulfate than the classical sex-linked cases. Both forms were shown to excrete a fraction of chondroitin sulfate B that would precipitate as the calcium salt with 10% alcohol which is in contrast to the behavior of this substance isolated from other sources. An atypical “adult form” (autosomal recessive) was shown to excrete chondroitin sulfate B and heparitin sulfate in excess, but no fraction of con-droitin sulfate B was obtained that would precipitate as the calcium salt with 10% alcohol. Another atypical form, which shows primarily degeneration associated with the nervous system, was shown to be inherited in an autosomal recessive manner and to excrete only heparitin sulfate in excess. The authors extend their thanks to Dr. H. Scheie for allowing them to examine urine samples from 2 of his collected cases of atypical Hurlers syndrome to Dr. Edmond Burke and Miss Myrna Sproul for help in obtaining patients with this disease, and to Douglas M. Brown, Univ. of Utah College of Med. for molecular weight determinations. Also we are very gratefuly to the families involved who helped so graciously during this study. Finally, but not least, we thank Miss Kathryn Seymour for excellent technical assistance.

Morquio's disease: An abnormality of mucopolysaccharide metabolism

Two siblings with the clinical and radiographic manifestations of Morquios disease have been found to excrete keratosulfate, a mucopolysaccharide not found in the urine of normal people or in patients with Hurlers disease. Although not usually listed as characteristics of Morquios disease, it is suggested that corneal opacities, Reilly granules in the leukocytes, and abnormal mucopolysaccharides in the urine may be found in a high percentage of these patients if they are looked for carefully. An 8-month-old baby is presented who has no clinical signs of Morquios disease, but who was found to have diagnostic radiographic findings, Reilly bodies in leukocytes, and keratosulfate in the urine.

The enzymatic degradation of heparitin sulfate. II. Isolation and characterization of non-sulfated oligosaccharides.

Abstract A non-sulfated, N-acetylated disaccharide was isolated from hepatirin sulfate after enzymatic digestion. This compound containing α, β unsaturated uronic acid is an isomer of a disaccharide obtained previously from hyaluronic acid. It contains an α- d -(1 → 4) rather than the β- d -(1 → 3) linkage found in the hyaluronate compound. The isolation of another non-sulfated, N-acetylated compound, most likely a tetrasaccharide, indicates that at least two or more N-acetylated units occur in sequence in the polysaccharide. The linkages in the non-sulfated portion of heparitin sulfate appear to be mainly α- d -(1 → 4) .