Energetics of hydrogen bond switch, residue burial and cavity analysis reveals molecular basis of improved heparin binding to antithrombin

Antithrombin III (ATIII) is the main inhibitor of the coagulation proteases like factor Xa and thrombin. Anticoagulant activity of ATIII is increased by several thousand folds when activated by vascular wall heparan sulfate proteoglycans (HSPGs) and pharmaceutical heparins. ATIII isoforms in human plasma, alpha-ATIII and beta-ATIII differ in the amount of glycosylation which is the basis of differences in their heparin binding affinity and function. Crystal structures and site directed mutagenesis studies have mapped the heparin binding site in ATIII, however the hydrogen bond switch and energetics of interaction during the course of heparin dependent conformational change remains largely unclear. An analysis of heparin bound conformational states of ATIII using PEARLS software showed that in heparin bound intermediate state, Arg 47 and Arg 13 residues make hydrogen bonds with heparin but in the activated conformation Lys 11 and Lys 114 have more hydrogen bond interactions. In the protease bound-antithrombin-pentasaccharide complex Lys 114, Pro 12 and Lys 125 form important hydrogen bonding interactions. The results showed that A-helix and N-terminal end residues are more important in the initial interactions but D-helix is more important during the latter stage of conformational activation and during the process of protease inhibition. We carried out the residue wise Accessible Surface Area (ASA) analysis of alpha and beta ATIII native states and the results indicated major differences in burial of residues from Ser 112 to Ser 116 towards the N-terminal end. This region is involved in the P-helix formation on account of heparin binding. A cavity analysis showed a progressively larger cavity formation during activation in the region just adjacent to the heparin binding site towards the C-terminal end. We hypothesize that during the process of conformational change after heparin binding beta form of antithrombin has low energy barrier to form D-helix extension toward N and C-terminal end as compared to alpha isoform.     

A peptide model for the heparin binding site of antithrombin III.

A peptide model for the heparin binding site of antithrombin III (ATIII) was synthesized to elucidate the structural consequences of heparin binding. This peptide [ATIII(123-139)] and a sequence-permuted analogue (ATIII random) showed similar conformational behavior (as analyzed by circular dichroism spectroscopy) in aqueous and organic media. In the presence of heparin, however, the peptide ATIII(123-139) assumed a stable conformation, whereas peptide ATIII random did not. Complex formation was saturable and sensitive to salt. The ATIII(123-139)-heparin complex contained beta-structure, rather than helical structure. This finding is incompatible with current models of heparin binding and suggests that heparin binding may induce nonnative structures at the binding site which could, in turn, lead to activation of ATIII. The peptide ATIII(123-139) was able to inhibit the binding of ATIII by heparin, consistent with the notion that this peptide may be a model for the heparin binding site.     

Important role of arginine 129 in heparin-binding site of antithrombin III. Identification of a novel mutation arginine 129 to glutamine

An hereditary abnormal antithrombin III (ATIII Geneva) with defective heparin cofactor activity was characterized by DNA single strand amplification and subsequent direct sequencing. ATIII Geneva was found to have a G to A transition in Exon IIIa leading to an Arg-129 to Gln mutation. This amino acid is part of the ATIII region comprising residues 114-154, which contains the highest proportion of basic residues (Arg or Lys), and is known from chemical modification studies to be involved in heparin binding. The variant protein did not bind heparin-Sepharose and was isolated from the propositus plasma by immunoaffinity chromatography. High affinity (for ATIII) heparin had only a minimal effect on thrombin and activated factor X inhibition by the purified abnormal ATIII. Taken together, these results demonstrate an important role for Arg-129 in the binding and interaction of ATIII with heparin of high affinity. We propose that a cooperation between Lys-125, Arg-129, Lys-136, and Arg-47 exposed at the surface of the inhibitor allows the binding of the essential pentasaccharide domain of heparin which is specific for the ATIII interaction.     

Identification of a heparin binding domain in the N-terminal cleavage site of pro-islet amyloid polypeptide. Implications for islet amyloid formation

Islet amyloid deposits are a characteristic pathologic lesion of the pancreas in type 2 diabetes and are composed primarily of the islet beta cell peptide islet amyloid polypeptide (IAPP or amylin) as well as the basement membrane heparan sulfate proteoglycan perlecan. Impaired processing of the IAPP precursor has been implicated in the mechanism of islet amyloid formation. The N- and C-terminal cleavage sites where pro-IAPP is processed by prohormone convertases contain a series of basic amino acid residues that we hypothesized may interact with heparan sulfate proteoglycans. This possibility was tested using affinity chromatography by applying synthetic fragments of pro-IAPP to heparin-agarose and heparan sulfate-Sepharose. An N-terminal human pro-IAPP fragment (residues 1-30) was retained by both heparin-agarose and heparan sulfate-Sepharose, eluting at 0.18 m NaCl at pH 7.5. Substitution of alanine residues for two basic residues in the N-terminal cleavage site abolished heparin and heparan sulfate binding activity. At pH 5.5, the affinity of the wild-type peptide for heparin/heparan sulfate was increased, implying a role for histidine residues at positions 6 and 28 of pro-IAPP. A C-terminal pro-IAPP fragment (residues 41-67) had no specific affinity for either heparin or heparan sulfate, and the N- or C-terminal fragments had only weak affinity for chondroitin sulfate. These data suggest that monomeric N-terminal human pro-IAPP contains a heparin binding domain that is lost during normal processing of pro-IAPP.     

A heparin binding site in antithrombin III. Identification, purification, and amino acid sequence

A heparin-binding peptide within antithrombin III (ATIII) was identified by digestion of ATIII with Staphylococcus aureus V8 protease followed by purification on reverse-phase high pressure liquid chromatography using a C-4 column matrix. The column fractions were assayed for their ability to bind heparin by ligand blotting with 125I-fluoresceinamine-heparin as previously described (Smith, J. W., and Knauer, D. J. (1987) Anal. Biochem. 160, 105-114). This analysis identified at least three fractions with heparin binding ability of which the peptide eluting at 25.4 min gave the strongest signal. Amino acid sequence analysis of this peptide gave a partially split sequence which was consistent with regions encompassing amino acids 89-96 and 114-156. These amino acids are present in a 1:1 molar ratio which is consistent with a disulfide linkage between Cys-95 and Cys-128. High affinity heparin competed more effectively for the binding of 125I-fluoresceinamine-heparin to this peptide than low affinity heparin. Chondroitin sulfate did not block the binding of 125I-fluoresceinamine-heparin to the peptide. These data strongly suggest that the isolated peptide represents a native heparin-binding region within intact ATIII. Computer generation of a plot of running charge density of ATIII confirms that the region encompassing amino acid residues 123-141 has the highest positive charge density within the molecule. A hydropathy plot of ATIII was generated using a method similar to that of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132). This plot indicates that amino acid residues 126-140 are exposed to the exterior surface of the molecule. Based on these data, we suggest that the region corresponding to amino acid residues 114-156 is a likely site for the physiological heparin-binding domain of ATIII. We also conclude that the proposed disulfide bridges within the protein are suspect and should be re-examined (Petersen, T. E., Dudek-Wojiechowska, G., Sottrup-Jensen, L., and Magnussun, S. (1979) in The Physiological Inhibitors of Coagulation and Fibrinolysis (Collen, D., Wiman, B., and Verstaeta, M., eds) pp. 43-54, Elsevier Scientific Publishing Co., Amsterdam).     

The heparin binding site of human extracellular-superoxide dismutase.

Extracellular-superoxide dismutase (EC-SOD) is a secretory glycoprotein that is major SOD isozyme in extracellular fluids. We revealed the possible structure of the carbohydrate chain of serum EC-SOD with the serial lectin affinity technique. The structure is a biantennary complex type with an internal fucose residue attached to asparagine-linked N-acetyl-D-glucosamine and with terminal sialic acid linked to N-acetyllactosamine. EC-SOD in plasma is heterogeneous with regard to heparin affinity and can be divided into three fractions: A, without affinity; B, with intermediate affinity; and C, with high affinity. It appeared that this heterogeneity is not dependent on the carbohydrate structure upon comparison of EC-SOD A, B, and C. No effect of the glycopeptidase F treatment of EC-SOD C on its heparin affinity supported the results. A previous report showed that both lysine and arginine residues probably at the C-terminal end, contribute to heparin binding. Recombinant EC-SOD C treated with trypsin or endoproteinase Lys C, which lost three lysine residues (Lys-211, Lys-212, and Lys-220) or one lysine residue (Lys-220) at the C-terminal end, had no or weak affinity for the heparin HPLC column, respectively. The proteinase-treated r-EC-SOD C also lost triple arginine residues which are adjacent to double lysine residues. These results suggest that the heparin-binding site may occur on a "cluster" of basic amino acids at the C-terminal end of EC-SOD C. EC-SOD is speculated to be primarily synthesized as type C, and types A and B are probably the result of secondary modifications. It appeared that the proteolytic cleavage of the exteriorized lysine- and arginine-rich C-terminal end in vivo is a more important contributory factor to the formation of EC-SOD B and/or EC-SOD A.     

Extension and structural variability of the antithrombin-binding sequence in heparin

Oligosaccharides with different affinities for antithrombin were isolated following partial deaminative cleavage of pig mucosal heparin with nitrous acid. The smallest high-affinity component obtained was previously identified as an octasaccharide with the predominant structure: (Formula: see text). The interaction of this octasaccharide, and of deca- and dodecasaccharides containing the same octasaccharide sequence, with antithrombin was studied by spectroscopic techniques. The near-ultraviolet difference spectra, circular dichroism spectra, and fluorescence enhancements induced by adding these oligosaccharides to antithrombin differed only slightly from the corresponding parameters measured in the presence of undegraded high-affinity heparin. Moreover, the binding constants obtained for the oligosaccharides and for high-affinity heparin were similar (1.0-2.9 X 10(7) M-1 at I = 0.3). In contrast, two hexasaccharides corresponding to units 1-6 and 3-8, respectively, of the above sequence showed about a 1000-fold lower affinity for antithrombin, and also induced considerably different spectral perturbations in antithrombin. Since the 1-6 hexasaccharide contains a reducing-terminal anhydromannose residue instead of the N-sulfated glucosamine unit 6 of the intact sequence, these results strongly support our previous conclusion that the N-sulfate group at position 6 is essential to the interaction with antithrombin. The low affinity of the hexasaccharide 3-8 provides further evidence that a pentasaccharide sequence 2-6 constitutes the actual antithrombin-binding region in the heparin molecule. Structural analysis of the various oligosaccharides revealed natural variants with an N-sulfate group substituted for the N-acetyl group at position 2. The preponderance of N-acetyl over N-sulfate groups at this position may be rationalized in terms of the mechanism of heparin biosynthesis, assuming that the D-gluco configuration of unit 3 is an essential feature of the antithrombin-binding region.     

Short heparin sequences spaced by glycol-split uronate residues are antagonists of fibroblast growth factor 2 and angiogenesis inhibitors

Fibroblast Growth Factor-2 (FGF2) is a major inducer of neovascularization (angiogenesis). Heparin activates FGF2 by favoring formation of ternary complexes with its cellular receptors (FGFRs). Controlled 2-O-desulfation followed by exhaustive periodate oxidation/borohydride reduction has been used to generate sulfation gaps within the prevalent heparin sequences, building-up arrays of pentasulfated trisaccharides (PST, consisting of a 2-O-sulfated iduronic acid flanked by two N,6-disulfated glucosamines) spaced by reduced, glycol-split uronic acid (sU) residues. The structure of the prevalent sequences of the novel heparin derivative has been confirmed by mono- and two-dimensional NMR analysis. NMR spin-lattice relaxation times (T2) and nuclear Overhauser effects suggest that the sU residues act as flexible joints between the PST sequences and cause a marked distortion of the chain conformation of heparin required for formation of ternary complexes. Since the splitting reaction also occurs at the level of the essential glucuronic acid residue of the active site for antithrombin, the heparin derivative has no anticoagulant activity. However, it fully retains the FGF2-binding ability of the original heparin, as shown by its capacity to protect FGF2 from trypsin cleavage and to prevent the formation of heparan sulfate proteoglycan (HSPG)/FGF2/FGFR1 ternary complexes. However, when compared to heparin it showed a reduced capacity to induce FGF2 dimerization and to favor the interaction of [125I]FGF2 with FGFR1 in HSPG-deficient, FGFR1-transfected CHO cells. Accordingly, it was more effective than heparin in inhibiting the mitogenic activity exerted by FGF2 in cultured endothelial cells. Finally, it inhibited angiogenesis in a chick embrio chorioallantoic membrane (CAM) assay in which heparin is inactive.     

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.     

Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy.

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123-G148, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L130-G148 and K136-G148 displayed attenuated heparin binding activities. In fact, K136-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K121-A134) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low microM concentrations of heparin induce significant alpha-helix conformation. K121-A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-ATIII interactions.     

Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS/heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS/heparin biosynthesis might be necessary.     

Preparation and anticoagulant activity of fully O-sulphonated glycosaminoglycans

Glycosaminoglycans including dermatan sulphate, hyaluronan, heparan sulphate and heparin were chemically modified by O-sulphonation. By altering the reaction conditions, products having a different degree of O-sulphonation could be obtained. Glycosaminoglycan derivatives were prepared having no free hydroxyl groups, with sulphoester group/disaccharide unit ratios of 4.0 for dermatan sulphate and hyaluronan, and sulphoester and sulphamide group/disaccharide unit ratios of 4.22 and 4.88 for heparan sulphate and heparin, respectively. 1H NMR spectroscopy showed that the fully O-sulphonated hyaluronan derivative had a glucuronate residue with an altered conformation. Since glycosaminiglycans and their derivatives are often used as anticoagulant/antithrombotic agents, their anti-amidolytic activities were determined. The anti-factor IIa activity of fully O-sulphonated dermatan sulphate, hyaluronan and heparan sulphate ranged from 40 to 80 units/mg, while no anti-factor Xa activity of the fully O-sulphonated glycosaminoglycans was detected. These values are lower than those reported for low-molecular-weight heparins and are consistent with the requirement of an antithrombin III pentasaccharide binding site for anti-factor Xa activity. Interestingly, the anti-factor Xa of heparin is lost by chemical O-sulphonation.     

Antithrombin III phenylalanines 122 and 121 contribute to its high affinity for heparin and its conformational activation

The dissociation equilibrium constant for heparin binding to antithrombin III (ATIII) is a measure of the cofactor's binding to and activation of the proteinase inhibitor, and its salt dependence indicates that ionic and non-ionic interactions contribute approximately 40 and approximately 60% of the binding free energy, respectively. We now report that phenylalanines 121 and 122 (Phe-121 and Phe-122) together contribute 43% of the total binding free energy and 77% of the energy of non-ionic binding interactions. The large contribution of these hydrophobic residues to the binding energy is mediated not by direct interactions with heparin, but indirectly, through contacts between their phenyl rings and the non-polar stems of positively charged heparin binding residues, whose terminal amino and guanidinium groups are thereby organized to form extensive and specific ionic and non-ionic contacts with the pentasaccharide. Investigation of the kinetics of heparin binding demonstrated that Phe-122 is critical for promoting a normal rate of conformational change and stabilizing AT*H, the high affinity-activated binary complex. Kinetic and structural considerations suggest that Phe-122 and Lys-114 act cooperatively through non-ionic interactions to promote P-helix formation and ATIII binding to the pentasaccharide. In summary, although hydrophobic residues Phe-122 and Phe-121 make minimal contact with the pentasaccharide, they play a critical role in heparin binding and activation of antithrombin by coordinating the P-helix-mediated conformational change and organizing an extensive network of ionic and non-ionic interactions between positively charged heparin binding site residues and the cofactor.     

Site-directed mutagenesis and molecular modeling identify a crucial amino acid in specifying the heparin affinity of FGF-1.

Heparin potentiates the mitogenic activity of FGF-1 by increasing the affinity for its receptor and by extending its biological half-life. During the course of labeling human FGF-1 with Na(125)I and chloramine T, it was observed that the protein lost its ability to bind to heparin. In contrast, bovine FGF-1 retained its heparin affinity even after iodination. To localize the region responsible for the lost heparin affinity, chimeric FGF-1 proteins were constructed from human and bovine FGF-1 expression constructs and tested for their heparin affinity after iodination. The results showed that the C-terminal region of human FGF-1 was responsible for the loss of heparin affinity. This region harbors a single tyrosine residue in human FGF-1 in contrast to a phenylalanine at this position in bovine FGF-1. Mutating this tyrosine residue in the human FGF-1 sequence to phenylalanine did not restore the heparin affinity of the iodinated protein. Likewise, changing the phenylalanine to tyrosine in the bovine FGF-1 did not reduce the ability of the iodinated protein to bind to heparin. In contrast, a mutant human FGF-1 that has cysteine-131 replaced with serine (C131S) was able to bind to heparin even after iodination while bovine FGF-1 (S131C) lost its binding affinity to heparin upon iodination. In addition, the human FGF-1 C131S mutant showed a decrease in homodimer formation when exposed to CuCl(2). Molecular modeling showed that the heparin-binding domain of FGF-1 includes cysteine-131 and that cysteine-131, upon oxidation to cysteic acid during the iodination procedures, would interact with lysine-126 and lysine-132. This interaction alters the conformation of the basic residues such that they no longer bind to heparin.     

Enhancement of neurite outgrowth-promoting activity by heparin derivatives in sodium chlorate-treated explant cultures of rat central neurons

Periodate-oxidized/borohydride-reduced 2-O-desulfated heparin (OR2DSH) was prepared using intact heparin from pig intestine as the starting material. Successive treatments of the heparin by oxidation with sodium periodate and reduction with sodium borohydride yielded periodate-oxidized/borohydride-reduced heparin (OR-heparin). Subsequent 2-O-desulfation of OR-heparin, according to a previously established method, yielded OR2DSH. Digestion of OR2DSH with heparitinases generated unsaturated disaccharides, comprising 86.5% DeltaDiHS-(6,N)S (DeltaUA1-->4GlcNS(6S)) and 13.5% DeltaDiHS-NS (DeltaUA1-->4GlcNS), as well as undigested oligosaccharides in which uronate moieties were derivatized by the cleavage of the covalent bond between the C-2 and C-3 positions by periodate-oxidation. The molecular mass of OR2DSH was determined to be 11 kDa, which is almost the same as those of other heparin derivatives such as 2-O-desulfated heparin (2DSH), 6-O-desulfated heparin (6DSH) and N-desulfated N-reacetylated heparin (NDSNAc-heparin). The ability of OR2DSH to enhance neurite outgrowth-promoting activity was evaluated using the explant culture of neocortical tissue from rat embryo in which endogenous heparan sulfate at the cell surface lost substantial numbers of sulfate groups by the action of 40 micro M sodium chlorate. The maximum activity of OR2DSH (29.7%) was achieved at 10 micro g/ml, and those of OR-heparin (21.7%), 2DSH (18.7%) and intact heparin (16.3%) were 100 micro g/ml, whereas that of NDSNAc-heparin (16.5%) was 1,000 micro g/ml. Completely 6-O-desulfated heparin (100:6DSH) exhibited very weak activity (3.3%) at 1,000 micro g/ml. These results suggest that the potency of OR2DSH to enhance neurite outgrowth-promoting activity is exerted synergetically by two different components in OR2DSH, i.e., the IdoA alpha1-->4GlcNS(6S) unit, which contains 6-O- and 2-N-sulfate groups, and the uronate moiety in which the covalent bond between C-2 and C-3 is cleaved, although the mode of action remains to be clarified.     

Search for the heparin antithrombin III-binding site precursor.

The last step of heparin biosynthesis is thought to involve the action of 3-O-sulfotransferase resulting in the formation of an antithrombin III (ATIII) binding site required for heparin's anticoagulant activity. The isolation of a significant fraction of heparin chains without antithrombin III-binding sites and having low affinity for ATIII suggests the presence of a precursor site, lacking the 3-O-sulfate group. Porcine mucosal heparin was depolymerized into a mixture of oligosaccharides using heparin lyase. One of these oligosaccharides was derived from heparin's ATIII-binding site. In an effort to find the ATIII-binding site precursor, the structures of several minor oligosaccharides were determined. A greater than 90% recovery of oligosaccharides (on a mole and weight basis) was obtained for both unfractionated and affinity-fractionated heparins. An oligosaccharide arising from the ATIII-binding site precursor was found that comprised only 0.8 mol % of the oligosaccharide product mixture. This oligosaccharide was only slightly enriched in heparin having a low affinity for ATIII and only slightly disenriched in high affinity heparin. The small number of these ATIII-binding site precursors, found in unfractionated and fractionated heparins, suggests the existence of a low ATIII affinity heparin may not simply be the result of the incomplete action of 3-O-sulfotransferase in the final step in heparin biosynthesis. Rather these data suggest that some earlier step, involved in the formation of placement of these precursor sites, may be primarily responsible for high and low ATIII affinity heparins.     

Human ficolin-2 recognition versatility extended: An update on the binding of ficolin-2 to sulfated/phosphated carbohydrates

Ficolin-2 has been reported to bind to DNA and heparin, but the mechanism involved has not been thoroughly investigated. X-ray studies of the ficolin-2 fibrinogen-like domain in complex with several new ligands now show that sulfate and phosphate groups are prone to bind to the S3 binding site of the protein. Composed of Arg132, Asp133, Thr136 and Lys221, the S3 site was previously shown to mainly bind N-acetyl groups. Furthermore, DNA and heparin compete for binding to ficolin-2. Mutagenesis studies reveal that Arg132, and to a lesser extent Asp133, are important for this binding property. The versatility of the S3 site in binding N-acetyl, sulfate and phosphate groups is discussed through comparisons with homologous fibrinogen-like recognition proteins.     

Structure and activity of a unique heparin-derived hexasaccharide

A hexasaccharide representing a major sequence in porcine mucosal heparin has been enzymatically prepared from heparin. Its structure was determined by an integrated approach using chemical, enzymatic, and spectroscopic methods. Two-dimensional 1H homonuclear COSY, C-H correlation NMR, and selective irradiation were used to assign many of the NMR resonances. In addition, new techniques including sulfate determination by ion chromatography and Fourier transform IR and californium plasma desorption mass spectroscopy have been applied, resulting in an unambiguous structural assignment of delta IdoAp2S(1----4)-alpha-D-GlcNp2S6S(1----4)-alpha-L-IdoAp++ +(1----4)-alpha-D-GlcNA cp6S-(1----4)-beta-D-GlcAp(1----4)-alpha-D-GlcNp2S3S6S (where delta IdoA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, p represents pyranose, and GlcA and IdoA represent glucuronic and iduronic acid). This hexasaccharide contains a portion of the antithrombin III-binding site and has a Kd of 4 X 10(-5) M. Unlike other small heparin oligosaccharides, which are specific for coagulation factor Xa, it inhibits both factors IIa and Xa equally through antithrombin III. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor II to inhibit thrombin (factor IIa) and shows over half of heparin's heparin cofactor II-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin III, and heparin cofactor II.     

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.