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Papain is considered to be the archetype of cysteine proteinases. The interaction of heparin and other glycosaminoglycans with papain may be representative of many mammalian cysteine proteinase-glycosaminoglycan interactions that can regulate the function of this class of proteinases in vivo. The conformational changes in papain structure due to glycosaminoglycan interaction were studied by circular dichroism spectroscopy, and the changes in enzyme behavior were studied by kinetic analysis, monitored with fluorogenic substrate. The presence of heparin significantly increases the alpha-helix content of papain. Heparin binding to papain was demonstrated by affinity chromatography and shown to be mediated by electrostatic interactions. The incubation of papain with heparin promoted a powerful increase in the affinity of the enzyme for the substrate. In order to probe the glycosaminoglycan structure requirements for the papain interaction, the effects of two other glycosaminoglycans were tested. Like heparin, heparan sulfate, to a lesser degree, was able to decrease the papain substrate affinity, and it simultaneously induced alpha-helix structure in papain. On the other hand, dermatan sulfate was not able to decrease the substrate affinity and did not induce alpha-helix structure in papain. Heparin stabilizes the papain structure and thereby its activity at alkaline pH.

Cell-fibronectin interactions, mediated through several different receptors, have been implicated in a wide variety of cellular properties. Among the cell surface receptors for fibronectin, integrins are the best characterized, particularly the prototype alpha5beta1 integrin. Using [125I]iodine cell surface labeling or metabolic radiolabeling with sodium [35S]sulfate, we identified alpha5beta1 integrin as the only sulfated integrin among beta1 integrin heterodimers expressed by the human melanoma cell line Mel-85. This facultative sulfation was confirmed not only by immunoprecipitation reactions using specific monoclonal antibodies but also by fibronectin affinity chromatography, two-dimensional electrophoresis, and chemical reduction. The covalent nature of alpha5beta1 integrin sulfation was evidenced by its resistance to treatments with high ionic, chaotrophic, and denaturing agents such as 4 M NaCl, 4 M MgCl2, 8 M urea, and 6 M guanidine HCl. Based on deglycosylation procedures as chemical beta-elimination, proteinase K digestion, and susceptibility to glycosaminoglycan lyases (chondroitinase ABC and heparitinases I and II), it was demonstrated that the alpha5beta1 heterodimer and alpha5 and beta1 integrin subunits were proteoglycans. The importance of alpha5beta1 sulfation was strengthened by the finding that this molecule is also sulfated in MG-63 (human osteosarcoma) and HCT-8 (human colon adenocarcinoma) cells.

The isolation and some structural features of heparan sulfates and chondroitin sulfates from three species of molluscs (Pomacea sp., Tagelus gibbus, and Anomalocardia brasiliana) are reported. It is shown that heparan sulfates with structural similarities to the ones found in mammals are present in the three molluscs analyzed. All the heparan sulfates were degraded by heparitinases I and II to four distinct unsaturated disaccharides with the same properties as the ones present in heparan sulfates of mammalian origin. This suggests that these four disaccharide units are maintained through the evolution. Furthermore, the proportion of these units varied in the heparan sulfates according to the species of origin. The chondroitin sulfates, on the other hand, exhibit different structural features according to the species of origin. For instance, by the action of chondroitinase AC, 4- and nonsulfated disaccharides are produced from Pomacea chondroitin, whereas 4- and 6-sulfated disaccharides are formed from Tagelus and Anomalocardia. The possible role of these compounds in cell recognition and/or adhesiveness is discussed in view of the present findings.

The heparin content of genetically selected mice with high and low antibody response to bacterial antigens is reported. An inverse relationship between antibody titres and concentration of heparin was observed for both male and female mice. The lower-antibody-responder line contains twice as much heparin as the higher-responder ones. Furthermore, the female mice also contained twice as much heparin as the male mice. Genetic analysis of the parental and interline hybrids has shown a partial dominance for the character ‘heparin content’ in favour of the high-heparin phenotype and this character appears to be subjected to polygenic control. The possible biological role of heparin and/or mast cells in the surveillance of the organism against some pathogens is discussed in the light of these and other findings.

The lumen of the Golgi apparatus is the subcellular site where galactose is transferred, from UDP-galactose, to the oligosaccharide chains of glycoproteins, glycolipids, and proteoglycans. The nucleotide sugar, which is synthesized in the cytosol, must first be transported into the Golgi lumen by a specific UDP-galactose transporter. Previously, a mutant polarized epithelial cell (MDCKII-RCAr) with a 2% residual rate of transport of UDP-galactose into the lumen of Golgi vesicles was described (Brandli, A. W., Hansson, G. C., Rodriguez-Boulan, E., and Simons, K. (1988) J. Biol. Chem. 263, 16283-16290). The mutant has an enrichment in glucosyl ceramide and cell surface glycoconjugates bearing terminal N-acetylglucosamine, as well as a 75% reduction in sialylation of cell surface glycoproteins and glycosphingolipids. We have now studied the biosynthesis of galactose containing proteoglycans in this mutant and the corresponding parental cell line. Wild-type Madin-Darby canine kidney cells synthesize significant amounts of chondroitin sulfate, heparan sulfate, and keratan sulfate, while the above mutant synthesizes chondroitin sulfate and heparan sulfate but not keratan sulfate, the only proteoglycan containing galactose in its glycosaminoglycan polymer. The mutant also synthesizes chondroitin 6-sulfate rather than only chondroitin 4-sulfate as wild-type cells. Together, the above results demonstrate that the Golgi membrane UDP-galactose transporter is rate-limiting in the supply of UDP-galactose into the Golgi lumen; this in turn results in selective galactosylation of macromolecules. Apparently, the Km for galactosyltransferases involved in the synthesis of linkage regions of heparan sulfate and chondroitin sulfate are significantly lower than those participating in the synthesis of keratan sulfate polymer, glycoproteins, and glycolipids. The results also suggest that the 6-O-sulfotransferases, in the absence of their natural substrates (keratan sulfate) may catalyze the sulfation of chondroitin 4-sulfate as alternative substrate.

The structure of the glycosaminoglycan chain of a heparan sulfate proteoglycan isolated from the conditioned medium of an endothelial cell line has been analyzed by using various degradative enzymes (heparitinase I, heparitinase II, heparinase, glycuronidase, sulfatases) from Flavobacterium heparinum. This proteoglycan inhibits the thromboplastin-activated pathway of coagulation; as a consequence, the catalytic conversion of prothrombin to thrombin is arrested. Heparitinase I (EC 4.2.2.8), an enzyme with specificity restricted to the heparan sulfate portion of the polysaccharide, releases fragments with the electrophoretic mobility and the structure of heparin. Conversely, an assessment of the size and distribution of the heparan sulfate regions has been provided by the use of heparinase (EC 4.2.2.7), which, by degrading the heparin sections of the chain, releases two segments that exhibit the structure of heparan sulfate. One of these segments is attached to the protein core. On the basis of these findings, the heparan sulfate chain can be defined as a copolymer containing heparin regions in its structure. The combined use of these enzymes has made it possible to establish the disaccharide sequence of parts of the glycosaminoglycan moiety of this proteoglycan.

The pharmacological activities of 14 heparins prepared from bovine and hog tissues and heparin fractions obtained by molecular sieving on sephacryl S-200 and antithrombin III affinity chromatography are reported. Two groups of heparins can be distinguished regarding the lipoprotein lipase releasing activity (LPL) and the activated partial thromboplastin time (APTT). Heparins prepared from liver, lung, pancreas and thymus have a ratio of LPL/APTT of 3.6, whereas heparins prepared from placenta and intestine show a ratio of only 2.1. The heparin APTT, LPL, antihemolytic, antifactor Xa and anticoagulant activities were partially resolved by fractionation on Sephacryl S-200 chromatography and antithrombin III affinity chromatography. These results suggest the presence of a mixture of heparins with different pharmacological activities in a commercial preparation.

Heparin with high anticoagulant activity was isolated from the two marine clam species Anomalocardia brasiliana and Tivela mactroides. A large portion of the polysaccharide chains of both preparations bound with high affinity to immobilized antithrombin. Titrations monitored by tryptophan fluorescence showed that clam polysaccharide chains with Mr approximately 22,500 contained up to three binding sites for antithrombin and that the binding constants for the interaction of these chains with antithrombin were higher than those reported for mammalian heparin of comparable size. Structural analysis of clam heparin fractions and subfractions of clam heparin with differing affinity for immobilized antithrombin revealed the presence of large amounts (up to 25-30% of the total disaccharide units) of the 3-O-sulfated saccharide sequences (-GlcNSO3)-GlcA-GlcNSO3(3-OSO3)- and (-GlcNSO3)-GlcA-GlcNSO3(3,6-di-OSO3)-, previously identified as unique markers for the antithrombin-binding region of heparin. The content of these saccharide sequences was found to increase with increasing affinity of the parent polysaccharide for antithrombin. Structural analysis of the clam heparins also demonstrated the occurrence of a novel saccharide sequence, tentatively identified as (-GlcNSO3)-IdA-GlcNSO3(3,6-di-OSO3)-, that has not previously been found in heparin or related polysaccharides. The contents of this latter sequence, at most 3-4% of the total disaccharide units, showed no correlation with the affinity for antithrombin.