Putative Heparin-binding Site Research Articles

Structural and Functional Characterization of a Highly Specific Serpin in the Insect Innate Immunity

The Toll signaling pathway, an essential innate immune response in invertebrates, is mediated via the serine protease cascade. Once activated, the serine proteases are irreversibly inactivated by serine protease inhibitors (serpins). Recently, we identified three serpin-serine protease pairs that are directly involved in the regulation of Toll signaling cascade in a large beetle, Tenebrio molitor. Of these, the serpin SPN48 was cleaved by its target serine protease, Spätzle-processing enzyme, at a noncanonical P1 residue of the serpin's reactive center loop. To address this unique cleavage, we report the crystal structure of SPN48, revealing that SPN48 exhibits a native conformation of human antithrombin, where the reactive center loop is partially inserted into the center of the largest β-sheet of SPN48. The crystal structure also shows that SPN48 has a putative heparin-binding site that is distinct from those of the mammalian serpins. Ensuing biochemical studies demonstrate that heparin accelerates the inhibition of Spätzle-processing enzyme by a proximity effect in targeting the SPN48. Our finding provides the molecular mechanism of how serpins tightly regulate innate immune responses in invertebrates.

Structure of the micronemal protein 2 A/I domain from Toxoplasma gondii

Toxoplasma gondii is a widespread zoonotic pathogen capable of causing serious disease in humans and animals. As an obligate intracellular parasite, T. gondii relies on the orchestrated secretion of proteins from its apical complex organelles including the multimodular, transmembrane micronemal protein 2 (MIC2) that couples recognition of the host cell with cytoskeletal reorganization of the parasite to drive invasion. To probe the basis by which the von Willebrand Factor A (vWA)-Integrin like module of TgMIC2 engages the host cell, we solved the crystal structure of a truncated form of TgMIC2A/I (TgMIC2A/Ic) phased by iodide SIRAS and refined to a resolution of 2.05 Å. The TgMIC2A/Ic core is organized into a central twisted beta sheet flanked by α-helices consistent with a canonical vWA fold. A restricted basic patch serves as the putative heparin binding site, but no heparin binding was detected in native gel shift assays. Furthermore, no metal was observed in the metal ion dependent adhesion site (MIDAS). Structural overlays with homologous A/I domains reveal a divergent organization of the MIDAS β4-α4 loop in TgMIC2A/Ic, which is stabilized through the burial of Phe195 into a deep pocket formed by Gly185. Intriguingly, Gly185 appears to be unique among A/I domains to TgMIC2A/I suggesting that the divergent loop conformation may also be unique to TgMIC2A/I. Although lacking the C-terminal extension, the TgMIC2A/Ic structure reported here is the first of an A/I domain from an apicomplexan parasite and provides valuable insight into defining the molecular recognition of host cells by these widespread pathogens.

Multiple mechanisms for exogenous heparin modulation of vascular endothelial growth factor activity

Heparin and heparin-like molecules are known to modulate the cellular responses to vascular endothelial growth factor-A (VEGF-A). In this study, we investigated the likely mechanisms for heparin's influence on the biological activity of VEGF-A. Previous studies have shown that exogenous heparin's effects on the biological activity of VEGF-A are many and varied, in part due to the endogenous cell-surface heparan sulfates. To circumvent this problem, we used mutant endothelial cells lacking cell-surface heparan sulfates. We showed that VEGF-induced cellular responses are dependent in part on the presence of the heparan sulfates, and that exogenous heparin significantly augments VEGF's cellular effects especially when endogenous heparan sulfates are absent. Exogenous heparin was also found to play a cross-bridging role between VEGF-A(165) and putative heparin-binding sites within its cognate receptor, VEGFR2 when they were examined in isolation. The cross-bridging appears to be more dependent on molecular weight than on a specific heparin structure. This was confirmed by surface plasmon resonance binding studies using sugar chips immobilized with defined oligosaccharide structures, which showed that VEGF-A(165) binds to a relatively broad range of sulfated glycosaminoglycan structures. Finally, studies of the far-UV circular dichroism spectra of VEGF-A(165) showed that heparin can also modulate the conformation and secondary structure of the protein.

The Heparin Binding Site of Protein C Inhibitor Is Protease-dependent

Protein C inhibitor (PCI) is a member of the serpin family of protease inhibitors with many biological functions and broad inhibitory specificity. Its major targets in blood are thrombin and activated protein C (APC), and the inhibition of both enzymes can be accelerated by glycosaminoglycans, including heparin. Acceleration of thrombin and APC inhibition by PCI requires that both protease and inhibitor bind to the same heparin chain to form a bridged Michaelis complex. However, the position of the heparin binding site of APC is opposite to that of thrombin, and formation of the bridged complexes must require either radical reorientation of the proteases relative to PCI or alternate heparin binding modes for PCI. In this study, we investigate how heparin bridges thrombin and APC to PCI by determining the effect of mutations in and around the putative heparin binding site of PCI. We found that heparin binds PCI in a linear fashion along helix H to bridge thrombin, consistent with our recent crystal structure (3B9F), but that it must rotate by approximately 60 degrees to engage Arg-229 to bridge APC. To gain insight into the possible modes of heparin binding to PCI, we solved a crystal structure of cleaved PCI bound to an octasaccharide heparin fragment to 1.55 angstroms resolution. The structure reveals a binding mode across the N terminus of helix H to engage Arg-229 and align the heparin binding site of APC. A molecular model for the heparin-bridged PCI.APC complex was built based on mutagenesis and structural data.

Neuroadapted Yellow Fever Virus Strain 17D: a Charged Locus in Domain III of the E Protein Governs Heparin Binding Activity and Neuroinvasiveness in the SCID Mouse Model

A molecular clone of yellow fever virus (YFV) strain 17D was used to identify critical determinants of mouse neuroinvasiveness previously localized to domain III of the neuroadapted SPYF-MN virus envelope protein. Three candidate virulence substitutions (305F-->V, 326K-->E, and 380R-->T) were individually evaluated for their roles in this phenotype in a SCID mouse model. The virus containing a glutamic acid residue at position 326 of the envelope protein (326E) caused rapidly lethal encephalitis, with a mortality rate and average survival time resembling those of the parental SPYF-MN virus. Determinants at positions 380 (380T) and 305 (305V) did not independently affect neuroinvasiveness. Testing a panel of viruses with various amino acid substitutions at position 326 revealed that attenuation of neuroinvasiveness required a positively charged residue (lysine or arginine) at this position. Molecular-modeling studies suggest that residues 326 and 380 contribute to charge clusters on the lateral surface of domain III that constitute putative heparin binding sites, as confirmed by studies of heparin inhibition of plaque formation. The neuroinvasiveness of YFVs in the SCID model correlated inversely with sensitivity to heparin. These findings establish that residue 326 in domain III of the E protein is a critical determinant of YFV neuroinvasiveness in the SCID mouse model. Together with modeling of domain III from virulent YFV strains, the data suggest that heparin binding activity involving lysine at position 326 may be a modulator of YFV virulence phenotypes.

Internalization and degradation of heparin is not required for stimulus of heparan sulfate proteoglycan synthesis

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.

The cell surface receptor FGFRL1 forms constitutive dimers that promote cell adhesion

FGFRL1 is a novel member of the fibroblast growth factor (FGF) receptor family. Utilizing the FRET (fluorescence resonance energy transfer) technique, we demonstrate that FGFRL1 forms constitutive homodimers at cell surfaces. The formation of homodimers was verified by co-precipitation of differentially tagged FGFRL1 polypeptides from solution. If overexpressed in cultivated cells, FGFRL1 was found to be enriched at cell–cell contact sites. The extracellular domain of recombinant FGFRL1 promoted cell adhesion, but not cell spreading, when coated on plastic surfaces. Adhesion was mediated by heparan sulfate glycosaminoglycans located at the cell surface. It could specifically be blocked by addition of soluble heparin but not by addition of other glycosaminoglycans. When the amino acid sequence of the putative heparin-binding site was modified by in vitro mutagenesis, the resulting protein exhibited decreased affinity for heparin and reduced activity in the cell-binding assay. Moreover, a synthetic peptide corresponding to the heparin-binding site was able to neutralize the effect of heparin. With its dimeric structure and its adhesion promoting properties, FGFRL1 resembles the nectins, a family of cell adhesion molecules found at cell–cell junctions.

Angiopoietin-Like Protein3 Regulates Plasma HDL Cholesterol Through Suppression of Endothelial Lipase

A low level of high-density lipoprotein (HDL) in plasma has been recognized as an aspect of metabolic syndrome and as a crucial risk factor of cardiovascular events. However, the physiological regulation of plasma HDL levels has not been completely defined. Current studies aim to reveal the contribution of angiopoietin-like protein3 (angptl3), previously known as a plasma suppressor of lipoprotein lipase, to HDL metabolism. Angptl3-deficient mice showed low plasma HDL cholesterol and HDL phospholipid (PL), and which were increased by ANGPTL3 supplementation via adenovirus. In vitro, ANGPTL3 inhibited the phospholipase activity of endothelial lipase (EL), which hydrolyzes HDL-PL and hence decreases plasma HDL levels, through a putative heparin-binding site in the N-terminal domain of ANGPTL3. Post-heparin plasma in Angptl3-knockout mice had higher phospholipase activity than did that in wild-type mice, suggesting that the activity of endogenous EL is elevated in Angptl3-deficient mice. Furthermore, we established an ELISA system for human ANGPTL3 and found that plasma ANGPTL3 levels significantly correlated with plasma HDL cholesterol and HDL-PL levels in human subjects. Angptl3 acts as an inhibitor of EL and may be involved in the regulation of plasma HDL cholesterol and HDL-PL levels in humans and rodents.

Computational Modeling of Glycosaminoglycan Protein Interactions of Collagen XI and Heparan Sulfate.

Interactions between glycosaminoglycans (GAGs), e.g. heparan sulfate, chondroitin sulfate, etc. and proteins are fundamental aspects of the extracellular matrix. Earlier models based on primary sequences of amino acids have successfully predicted some protein-GAG interactions, but models of the complex, folded three dimensional protein structures provide the opportunity for unexpected or unexplained interactions. Collagen XI has been shown to be a critical component in collagen fibril formation. It also contains four putative heparin-binding sites; one in the TSPN-like amino terminal domain, one in a region that is present only in certain isoforms, and two on the major triple helical domain. It is hypothesized that these putative binding sites mediate interactions between collagen XI-coated fibrils and the surrounding GAGs and contribute to the control of collagen fibril development. In order to move toward a process for three dimensional modeling of GAG-protein interactions and to better understand collagen XI interactions with the surrounding extracellular matrix, we have modeled, using Modeller v8.1 and various web-based threading and ab initio servers, each of the four putative heparin-binding domains. We utilized protein-ligand docking algorithms (e.g. Autodock) to determine the relative interaction energy and conformation between each putative binding site and a heparin molecule. The computational results were compared to in vitro binding data.

Humanized Recombinant Vaccinia Virus Complement Control Protein (hrVCP) with Three Amino Acid Changes, H98Y, E102K, and E120K Creating an Additional Putative Heparin Binding Site, Is 100‐fold More Active Than rVCP in Blocking Both Classical and Alternative Complement Pathways

Vaccinia virus complement control protein (VCP) is able to modulate the host complement system by regulating both pathways of complement activation. Efficient downregulation of complement activation depends on the ability of the regulatory protein to effectively bind the activated third (C3b) and fourth (C4b) complement components. Based on native crystallographic structure, molecular modeling, and sequence alignment with other Orthopoxviral complement control proteins (CCPs) and their host homologs, putative sites have been found on VCP as contact points for C3b/C4b. Here, we report that using site-directed mutagenesis, modified proteins have been generated. In addition, we report that the generated modified proteins with postulated contact point substitutions have shown greater ability to regulate both the classical and the alternative pathways of complement activation than the recombinant Western Reserve VCP, with one modified protein showing nearly 100-fold more potency in regulating both complement activation pathways independently. The augmented in vitro inhibitory activity of the modified protein together with the newly created putative heparin binding site suggests its promising potential as a competent therapeutic agent in modulating various complement-mediated ailments, for example, traumatic brain injury, Alzheimer's disease, rheumatoid arthritis, multiple organ dysfunction syndrome, reperfusion injury, and xenorejection.

Mapping the functional domains of human transcobalamin using monoclonal antibodies

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.

Solution Structure and Heparin Interaction of Human Hepatoma-derived Growth Factor

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a new protein family that has been implicated in nephrogenesis, tumorigenesis, vascular development, cell proliferation, and transcriptional activation. All HRPs share a conserved N-terminal homologous to the amino terminus of HDGF (HATH) domain, but vary significantly in the C-terminal region. Here, we show that in solution the N and C termini of human HDGF form two structurally independent domains. The 100 amino acid residue N-terminal HATH domain is well-structured while the 140 amino acid residue C-terminal domain is disordered. We determined the solution structure of the HATH domain by NMR. The core structure of the HATH domain is a five-stranded beta-barrel followed by two alpha-helices, similar to those of PWWP domains of known structures. Surface plasmon resonance results showed that the HATH domain is primarily responsible for heparin binding. On the basis of the chemical shift perturbation induced by binding of heparin-derived hexasaccharide, we identified a prominent, highly positively charged region as the putative heparin-binding site. Sequence comparison and structure prediction suggest that all HRPs are likely to adapt a similar modular structure.

Structural and functional dissection of the adhesive domains of Plasmodium falciparum thrombospondin-related anonymous protein (TRAP).

TRAP (thrombospondin-related anonymous protein) is a sporozoite surface protein that plays a central role in hepatocyte invasion. We have developed procedures for recombinant production of the entire ECD (extracellular domain) and A domain of TRAP using bacterial- and baculovirus-expression systems respectively. The ECD and A domain were purified to homogeneity and migrated on gel-filtration columns as non-aggregated, monomeric proteins. These adhesive modules bound to HepG2 cells in a dose-dependent and bivalent cation-independent manner. The binding of ECD and the A domain to HepG2 cells was inhibited poorly by an excess of sulphatide analogues, suggesting the presence of as yet unidentified receptors for the A domain on hepatocytes. Using surface-plasmon-resonance-based sensor technology (Biacore), we demonstrate that TRAP ECD has higher affinity for heparin (K(D)=40 nM) compared with the A domain (K(D)=79 nM). We also present a three-dimensional structure of the A domain based on the crystal structure of the homologous von Willebrand factor A1 domain. The TRAP A domain shows two spatially distinct ligand-binding surfaces. One surface on the A domain contains the MIDAS (metal-ion-dependent adhesion site) motif, where point mutations of Thr131 and Asp162 correlate with impairment of cell infectivity by sporozoites. The other surface contains a putative heparin-binding site and consists of a basic residue cluster. Our studies suggest that TRAP interacts with multiple receptors during the hepatocyte invasion process. Our results also pave the way for inclusion of these high-quality recombinant TRAP domains in subunit-based vaccines against malaria.

Functional Domains of a Pore-forming Cardiotoxic Protein, Volvatoxin A2

Volvatoxin A2 (VVA2), a novel pore-forming cardiotoxic protein was isolated from the mushroom Volvariella volvacea. We identified an N-terminal fragment (NTF) (1-127 residues) of VVA2 as a domain for oligomerization by limited tryptic digestion. On preincubation of NTF with VVA2, NTF was found to inhibit VVA2 hemolytic activity by inducing VVA2 oligomerization in the solution in the same manner as liposomes. By site-directed mutagenesis, the amphipathic alpha-helix B of NTF or VVA2 was shown to be indispensable for its biological functions. Interestingly, at a molar ratio of recombinant NTF (reNTF)/VVA2 as low as 0.01, reNTF was able to inhibit VVA2 hemolytic activity and induce VVA2 oligomerization. This indicates that reNTF can trigger VVA2 oligomerization by a seeding effect. Furthermore, the recombinant C-terminal fragment (128-199 residues) was found to be a functional domain that mediates the membrane binding of VVA2. We found a fragment localized at the C-terminal half of VVA2 containing beta6, -7, and -8, which is protected from protease digestion because of its insertion of a membrane. We also identified a putative heparin binding site (HBS) located in the VVA2 C terminus (166-194 residues), which was conserved among 10 kinds of snake venom cardiotoxins. VVA2 or the reHBS fragment was shown to interact with sulfated glycoaminoglycans by affinity column chromatography. The finding of a higher number of glycoaminoglycans in the membrane of cardiac myocytes suggested that they could be the specific membrane target for VVA2. Taken together, these findings indicate that VVA2 contains two functional domains, NTF and CTF. The NTF domain is responsible for VVA2 oligomerization and the CTF domain for membrane binding and insertion. Our results support a model whereby the formation of VVA2 oligomeric pre-pore complexes precedes their membrane insertion.

Conserved surface-exposed K/R-X-K/R motifs and net positive charge on poxvirus complement control proteins serve as putative heparin binding sites and contribute to inhibition of molecular interactions with human endothelial cells: a novel mechanism for evasion of host defense.

Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP-through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells-is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Molecular Features of the Collagen V Heparin Binding Site

A heparin binding region is known to be present within the triple helical part of the alpha1(V) chain. Here we show that a recombinant alpha1(V) fragment (Ile824 to Pro950), referred to as HepV, is sufficient for heparin binding at physiological ionic strength. Both native individual alpha1(V) chains and HepV are eluted at identical NaCl concentrations (0.35 M) from a heparin-Sepharose column, and this binding can be inhibited specifically by the addition of free heparin or heparan sulfate. In contrast, a shorter 23-residue synthetic peptide, containing the putative heparin binding site in HepV, fails to bind heparin. Interestingly, HepV promotes cell attachment, and HepV-mediated adhesion is inhibited specifically by heparin or heparan sulfate, indicating that this region might behave as an adhesive binding site. The same site is equally functional on triple helical molecules as shown by heparin-gold labeling. However, the affinities for heparin of each of the collagen V molecular forms tested are different and increase with the number of alpha1(V) chains incorporated in the molecules. Molecular modeling of a sequence encompassing the putative HepV binding sequence region shows that all of the basic residues cluster on one side of the helical face. A highly positively charged ring around the molecule is thus particularly evident for the alpha1(V) homotrimer. This could strengthen its interaction with the anionic heparin molecules. We propose that a single heparin binding site is involved in heparin-related glycosaminoglycans-collagen V interactions, but the different affinities observed likely modulate cell and matrix interactions between collagen V and heparan sulfate proteoglycans in tissues.

Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites.

We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect on the in vitro biological activity of IL-2. In addition soluble IL-2 receptor alpha and beta polypeptides do not compete with heparin for the binding of IL-2. IL-2 bound by heparin is still recognized by two IL-2 specific monoclonal antibodies, 3H9 and H2-8, whose epitopes lie in the amino terminal region. Murine IL-2 unlike its human counterpart fails to bind to heparin. Human IL-2 analogs with single amino acid substitutions at positions Lys43, Thr51, and Gln126 analogs no longer bind to heparin. By contrast the Arg38Ala analog retains heparin full heparin binding activity. These experimental findings together with molecular modeling studies suggest two putative heparin binding sites on human IL-2, one involving four basic residues, Lys48, Lys49, Lys54, and His55, and the other being a discontinuous site comprising Lys43, Lys64, Arg81, and Arg83. Neither of these two clusters is completely conserved in murine IL-2. Overall our data suggest that the binding of human IL-2 to heparin and heparan sulfate does not interfere with IL-2/IL-2 receptor interactions. Therefore, binding to glycosaminoglycan may be a mechanism for retaining the cytokine in an active form close to its site of secretion in the tissue, thus favoring a paracrine role for IL-2.

The solution structure of the N-terminal domain of hepatocyte growth factor reveals a potential heparin-binding site.

Hepatocyte growth factor (HGF) is a multipotent growth factor that transduces a wide range of biological signals, including mitogenesis, motogenesis, and morphogenesis. The N-terminal (N) domain of HGF, containing a hairpin-loop region, is important for receptor binding and the potent biological activities of HGF. The N domain is also the primary binding site for heparin or heparan sulfate, which enhances, receptor/ligand oligomerization and modulates receptor-dependent mitogenesis. The rational design of artificial modulators of HGF signaling requires a detailed understanding of the structures of HGF and its receptor, as well as the role of heparin proteoglycan; this study represents the first step towards that goal. We report here a high-resolution structure of the N domain of HGF. This first structure of HGF reveals a novel folding topology with a distinct pattern of charge distribution and indicates a possible heparin-binding site. The hairpin-loop region of the N domain plays a major role in stabilizing the structure and contributes to a putative heparin-binding site, which explains why it is required for biological functions. These results suggest several basic and/or polar residues that may be important for use in further mutational studies of heparin binding.

Effect of Individual Carbohydrate Chains of Recombinant Antithrombin on Heparin Affinity and on the Generation of Glycoforms Differing in Heparin Affinity

Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin, were previously shown to be expressed in BHK or CHO mammalian cell lines (I. Björk,et al.,1992,Biochem. J.286, 793–800; B. Fanet al.,1993,J. Biol. Chem.268, 17588–17596). To determine the source of the glycosylation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 96, 135, 155, and 192, was mutated to Gln to block glycosylation at these sites. Heparin-agarose chromatography of the four antithrombin variants revealed that Gln 96, Gln 135, and Gln 192 variants still displayed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gln 155 variant showed only a single functional high heparin affinity form. These results demonstrate that heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform. Analysis of heparin binding to the higher heparin affinity forms of the four variants showed that all exhibited increased heparin affinities of two- to sevenfold compared to wild-type higher heparin affinity form or to plasma antithrombin, with the Gln 135 variant showing the largest effect on this affinity. The extent of heparin-affinity enhancement was correlated with the distance of the mutated glycosylation site to the putative heparin-binding site in the X-ray structure of antithrombin. All variants displayed normal kinetics of thrombin inhibition in the absence and presence of saturating heparin, indicating that the carbohydrate chains solely affected heparin binding and not heparin-activation or proteinase-binding functions. These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect heparin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin.

Molecular cloning and sequence analysis of the mouse protein C inhibitor gene

The gene encoding mouse protein C inhibitor ( mPCI) was isolated and its nucleotide sequence determined. Alignment of the genomic sequence with that of a cDNA obtained from mouse testis revealed that the mPCI gene (like the human counterpart) is composed of five exons and four introns with highly conserved exon/intron boundaries. It encodes a pre-polypeptide of 405 amino acids, which shows 63% identity with human PCI (hPCI). The putative reactive site is identical to that of hPCI from P5 to P3′, suggesting a similar protease specificity. Also the putative heparin binding sites and `hinge' regions are highly homologous in mouse and hPCI.