Expression of C1 esterase inhibitor by the baculovirus expression vector system: preparation, purification, and characterization.

C1 esterase inhibitor (C1INH) is an important regulator of the classical complement pathway. Hereditary deficiency of C1INH causes angioedema of the skin, gut, and respiratory tissues that may be fatal. C1INH replacement therapy may be lifesaving for patients with this disorder. The objective of this study was to evaluate the use of the baculovirus expression vector system for mass producing biologically active human recombinant (rC1INH). A recombinant baculovirus was constructed coding the human native (nC1INH) sequence under control of the polyhedrin promoter. Spodoptera frugiperda Sf-9 insect cells were infected with this recombinant baculovirus in a medium-scale (10-L) bioreactor to produce rC1INH with a specific activity of 45 U/mg. Purification of rC1INH from the culture harvested at 60 h postinfection yielded 5.9 microg rC1INH/mL supernatant of a 75-kDa product with a specific activity of 31,000 U/mg purified rC1INH compared to 71,000 U/mg purified nC1INH from human serum using the same procedure. This rC1INH was about 25 kDa smaller than nC1INH, suggesting that Sf-9 cells express underglycosylated rC1INH. Glycan analysis showed that both N-glycan and O-glycan chains were present in rC1INH. The N-glycan chains, released using PNGaseF and fluorescently labeled, were analyzed using exoglycosidase treatment and capillary electrophoresis. Their high-mannose structure was consistent with the known failure of the insect cell glycosylation pathway to afford the fully elaborated biantennary structures found on human native nC1INH.

Effect of IGFBP-derived peptides on incorporation of(35)SO(4)into proteoglycans.

18 amino acid peptides from the C-terminal region of IGFBP-3, -5 (P3, P5), increased the incorporation of(35)SO(4)into proteoglycans in endothelial cells with greater stimulation in large vessel than microvessel cells. The homologous region of IGFBP-6 (P6) also stimulated sulfate uptake, but less potently than P3 and P5. P6 variants were synthesized with one or two amino acids changed to the basic amino acid in the equivalent position of P3. The P6 variants with one additional basic amino acid behaved similarly to P6. The P6 mutant with two altered amino acids was equipotent to P3. P3F, a scrambled version of P3 was less effective than P3. P3, P5, P6, P3F and all P6 variants all stimulated glucose uptake, which occurred only in microvessel cells. P1, P2, P4, and equimolar intact IGFBP-3 stimulated neither glucose uptake nor sulfate incorporation. Thus, C-terminal basic portions of IGFBP-3, -5 and -6 alter two specific functions of endothelial cells with sufficient differences to suggest mediation by distinct mechanisms.

Heparin binding and augmentation of C1 inhibitor activity.

Heparin and other glycosaminoglycans have profound activity in vitro on the regulation of complement activity. The studies reported here examined the mechanism whereby heparin enhances C1 esterase inhibitor (C1INH) activity on C1 esterase (C1). The interaction of heparin and heparan sulfate with C1INH was first examined using surface plasmon resonance. Heparin was immobilized on a biosensor chip in two orientations, at its reducing end and in midchain, and heparan sulfate was immobilized at its reducing end. Heparin immobilized at its reducing end interacted with C1INH, giving an association constant (Ka) value of 1.43 x 10(7) M-1, whereas heparin immobilized in midchain afforded a Ka value of 7 x 10(6) M-1. No interaction between C1INH and heparan sulfate could be observed. Next, the augmentation of C1INH by heparin (Mr (av) 13,000), low-molecular-weight (LMW) heparin (Mr (av) 5000), and heparan sulfate (Mr (av) 11,000) was determined. C1INH alone was at least 10, 000 times more active in inhibiting fluid phase C1 compared with erythrocyte-bound C1 (EAC1). When C1 was in the fluid phase, both heparin and LMW heparin were relatively ineffective at augmenting C1INH activity on C1. In contrast, when C1 was present as EAC1, heparin augmented C1INH activity at all C1INH concentrations examined and LMW heparin was up to 1.3 times more effective than heparin. This augmentation only occurred when both C1INH and heparin were present together with the EAC1. Hence, although surface plasmon resonance shows that heparin binds to C1INH, heparin augmentation of C1INH activity appears to require a terniary complex in which cell bound C1 interacts with both heparin and C1INH. This is the first report of LMW heparin augmenting C1INH activity. Heparan sulfate neither interacted with C1INH nor did it augment C1INH activity.

Pattern and spacing of basic amino acids in heparin binding sites.

Glycosaminoglycan (GAG)-protein interactions regulate a myriad of physiologic and pathologic processes, yet an understanding of how these molecules interact is lacking. The role of the pattern and spacing of basic amino acids (arginine (R) and lysine (K)) in heparin binding sites was investigated using peptide analogs as well as by examining known heparin binding sites. Peptides having the general structure R(n)W (n = 3-9, where tyrosine (W) was added for peptide detection) were synthesized and their interaction with heparin was determined by isothermal titration calorimetry. Binding affinity increased with increasing number of R residues. A 9-mer of R (R9W) bound as tightly to heparin as acidic fibroblast growth factor under physiologic conditions. Despite their high affinity for heparin, long stretches of basic amino acids are uncommon in heparin binding proteins. Known heparin binding sites most commonly contain single isolated basic amino acids separated by one nonbasic amino acid. Peptides having the structure, H3CCONH-GRRG(m)RRG(5-m)-CONH2 (denoted as the RRG(m)RR peptide series) and H3CCONH-GRRRG(m)RG(5-m)-CONH2 (denoted as the RRRG(m)R peptide series), where m = 0-5, were synthesized to test the hypothesis that the spacing of basic amino acids in heparin binding sites is optimally arranged to interact with different GAGs. The peptides, in both the -RRG(m)RR- and -RRRG(m)R- peptide series, when m = 0, bound most tightly with heparin, as measured by affinity chromatography. In contrast, the -RRG(m)RR-peptide series interacted most tightly with heparan sulfate when m = 0 or 1, whereas the -RRRG(m)R- peptide series bound tightest when m = 3. These results are consistent with our understanding of heparin and heparan sulfate structure. A highly sulfated GAG, such as heparin, interacts most tightly with peptides (or peptide sequences within proteins) containing a complementary binding site of high positive charge density. Heparan sulfate, having fewer and more highly spaced negatively charged groups, interacts most tightly with a complementary site on a peptide (or peptide sequences with proteins) that has more widely spaced cationic residues.

Structure-function relationships in the heparin-binding C-terminal region of insulin-like growth factor binding protein-3.

IGFBP-3 contains a carboxyterminal basic region which, when present as an isolated 18 amino acid peptide (P3), binds heparin, associates with cultured endothelial cells and stimulates glucose uptake. The P3 molecule has now been modified relative to charge, amino acid sequence and size to determine structure-function relationships relative to four properties of P3: affinity for heparin; inhibition of IGFBP-3 binding; stimulation of glucose uptake; and displacement of bFGF from the extracellular matrix of endothelial cells. Results indicate: (1) the presence or absence of heparin binding was concordant with the presence/absence of the other three properties; (2) the number of basic amino acids was an important, if not limiting, factor for each property; (3) the order of potency of the basic amino acids was arginine = lysine > > histidine; (4) the unrelated, basic protein, protamine, mimics all properties of P3; and (5) the putative consensus heparin-binding sequence of P3 was not essential for any of the P3 activities.

Importance of specific amino acids in protein binding sites for heparin and heparan sulfate.

Heparin and heparan sulfate bind a variety of proteins and peptides to regulate many biological activities. Past studies have examined a limited number of established heparin binding sites and have focused on basic amino acids when modeling binding site structural motifs. This study examines the prevalence of individual amino acids in peptides binding to heparin or heparan sulfate. A 7-mer random peptide library was synthesized using the 20 common amino acids. This 7-mer library was affinity separated using both heparin and heparan sulfate-Sepharose. Bound peptide populations were eluted with a salt step gradient (pH 7) and analysed for amino acid composition. Peptides released from heparin-Sepharose by 0.3 M NaCl were enriched in arginine, lysine, glycine and serine; and depleted in methionine and phenylalanine. In contrast, peptides released from heparan sulfate-Sepharose were enriched in arginine, glycine, serine, and proline (at 0.15 M NaCl). These peptides were depleted in histidine, isoleucine, methionine (not detectable) and phenylalanine. In the heparin binding sites of proteins, which have been published, the enriched amino acids were arginine, lysine and tyrosine. Depleted amino acids include aspartic acid, glutamic acid, glutamine, alanine, glycine, phenylalanine, serine, threonine and valine. This study demonstrates that heparin and heparan sulfate bind different populations of peptide sequences. The differences in amino acid composition indicate that the positive charge density and spacing requirements differ for peptides binding these two glycosaminoglycans.

Differences in the interaction of heparin with arginine and lysine and the importance of these basic amino acids in the binding of heparin to acidic fibroblast growth factor.

Although the interaction of proteins with glycosaminoglycans (GAGs) such as heparin are of great importance, the general structural requirements for protein- or peptide-GAG interaction have not been well characterized. Electrostatic interactions between sulfate and carboxylate groups on the GAG and basic residues in the protein or peptide dominate the interaction, but the thermodynamics of these electrostatic interactions have not been studied. Arginine residues occur frequently in the known heparin binding sites of proteins. Arginine is also more common than lysine in randomly synthesized 7-mer peptides that bind to immobilized heparin and heparan sulfate. We have used heparin affinity chromatography, equilibrium dialysis, and isothermal titration calorimetry techniques to further investigate these interactions. A 7-mer of arginine eluted from a heparin-affinity column at 0.82 M NaCl, whereas the analogous 7-mer of lysine eluted at 0.64 M. Similarly, the putative heparin binding site peptide (amino acid residues 110-130) from acidic fibroblast growth factor, which contained four lysine and two arginine residues, eluted at 0.50 M, whereas the analogous peptide with six lysine residues eluted at 0.41 M and one with six arginine residues eluted at 0.54 M. At 25 degrees C in 10 mM sodium phosphate, pH 7.4, carboxy and amino termini blocked arginine (blocked arginine) bound to heparin twice as tightly as blocked lysine as measured by equilibrium dialysis Similarly, at 30 degrees C in 10 mM sodium phosphate, pH 7.4, and in water, blocked arginine bound 2.5 times more tightly to anions in heparin than blocked lysine. Using titration calorimetry, the enthalpy of blocked arginine and lysine binding to heparin was 1.14 +/- 0.24 and 0.45 +/- 0.35 kJ/mol, respectively, under identical conditions. Our observations show that blocked arginine- and arginine-containing peptides bound more tightly to GAGs than the analogous lysine species and suggest that the difference was due to the intrinsic properties of the arginine and lysine side chains. The greater affinity of the guanidino cation for sulfate in GAGs is probably due to stronger hydrogen bonding and a more exothermic electrostatic interaction. This can be rationalized by soft acid, soft base concepts.

IGFBP-3 and IGFBP-5 association with endothelial cells: role of C-terminal heparin binding domain.

IGFBP-3 and IGFBP-5, but not the other 4 IGF binding proteins, specifically bound to endothelial cell (EC) monolayers. Charged compounds, such as heparin and heparan sulfate, competed for this binding. Of the 6 IGFBPs, IGFBP-3 and IGFBP-5 had the greatest heparin affinity. Peptides of 18 amino acids were synthesized, corresponding to a common basic region of IGFBP-3 (P3), IGFBP-5 and IGFBP-6 (P6) which contained a heparin binding sequence. P3 and P6 inhibited IGFBP-3 and -5 binding to endothelial cell monolayers and the peptides bound directly to EC extracellular matrix. This suggested that the C-terminal basic segment of IGFBP-3/-5 is important for the association of the binding protein with the EC monolayer.

Interaction of heparin with synthetic antithrombin III peptide analogues.

Heparin-binding proteins may contain specific patterns of basic amino acids, called consensus sequences, that interact with heparin. Small peptides were synthesized that contained consensus sequences (i.e. FAKLNCRLYRKANKSSK) or disrupted consensus sequences (i.e. K136-->A) based on the known sequence of antithrombin III (amino acid residues 123-139). These peptides were then examined in both competitive and non-competitive binding experiments using bioassays, fluorescence spectroscopy, affinity chromatography and n.m.r. spectroscopy. Both the consensus and disrupted-consensus peptide bound to heparin. Peptides with consensus sequences bound specifically to the pentasaccharide antithrombin III-binding site within heparin. In contrast, peptides with disrupted consensus sequences showed no specificity, binding to any sequence within heparin. Proton nuclear Overhauser enhancement spectroscopy demonstrated the proximity of leucine and tyrosine (within the consensus sequence) to the N-acetyl moiety found primarily within the pentasaccharide antithrombin III-binding site of heparin. This experiment confirmed the findings of the other techniques and helped to localize the binding sites in both peptides and heparin. A model is proposed for both specific and non-specific heparin interaction with consensus and disrupted-consensus peptides.

The Ac and Uq transposable element systems in maize: interactions among components.

Components of the Uq and Ac transposable element systems interact. A large sample of Ds-containing and ruq-containing alleles were tested against Uq and Ac. The Uq elements elicit a mutable response from only one of the classes of Ds elements (Ds1) in the Ac family. This response is similar to the response from ruq to Uq. In contrast, Ac elicits mutable responses from all Ds and ruq elements tested. This represents a lack of reciprocity of interaction for the components of the two elements, Ac and Uq. Further, two atypical Ac and Uq elements (Ac2 and Uq-Mn) were examined. All ruq and Ds elements tested respond to four doses of Ac2. (Responses to lower doses were not compared.) Only the ruq (Ds1) containing alleles respond to Uq-Mn. The other Ds containing alleles were nonresponsive. The finding of nonreciprocating interaction between components suggests a heterogeneous nature for transposable element systems in maize.