Modification of the active site of alkaline phosphatase by site-directed mutagenesis.

The catalytically essential amino acid in the active site of bacterial alkaline phosphatase (Ser-102) has been replaced with a cysteine by site-directed mutagenesis. The resulting thiol enzyme catalyzes the hydrolysis of a variety of phosphate monoesters. The rate-determining step of hydrolysis, however, is no longer the same for catalysis when the active protein nucleophile is changed from the hydroxyl of serine to the thiol of cysteine. Unlike the steady-state kinetics of native alkaline phosphatase, those of the mutant show sensitivity to the leaving group of the phosphate ester.

Pleiotropic effects of antithrombin strand 1C substitution mutations.

Six different substitution mutations were identified in four different amino acid residues of antithrombin strand 1C and the polypeptide leading into strand 4B (F402S, F402C, F402L, A404T, N405K, and P407T), and are responsible for functional antithrombin deficiency in seven independently ascertained kindreds (Rosny, Torino, Maisons-Laffitte, Paris 3, La Rochelle, Budapest 5, and Oslo) affected by venous thromboembolic disease. In all seven families, variant antithrombins with heparin-binding abnormalities were detected by crossed immunoelectrophoresis, and in six of the kindreds there was a reduced antigen concentration of plasma antithrombin. Two of the variant antithrombins, Rosny and Torino, were purified by heparin-Sepharose and immunoaffinity chromatography, and shown to have greatly reduced heparin cofactor and progressive inhibitor activities in vitro. The defective interactions of these mutants with thrombin may result from proximity of s1C to the reactive site, while reduced circulating levels may be related to s1C proximity to highly conserved internal beta strands, which contain elements proposed to influence serpin turnover and intracellular degradation. In contrast, s1C is spatially distant to the positively charged surface which forms the heparin binding site of antithrombin; altered heparin binding properties of s1C variants may therefore reflect conformational linkage between the reactive site and heparin binding regions of the molecule. This work demonstrates that point mutations in and immediately adjacent to strand 1C have multiple, or pleiotropic, effects on this serpin, leading ultimately to failure of its regulatory function.

Antithrombin Budapest 3. An antithrombin variant with reduced heparin affinity resulting from the substitution L99F.

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, CTC----TTC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT-binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution-induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.

Molecular genetic survey of 16 kindreds with hereditary antithrombin III deficiency.

Molecular genetic techniques were utilized to examine antithrombin III (ATIII) gene status in 16 independently ascertained kindreds with hereditary ATIII deficiency. In one of these families antithrombin III deficiency is caused by hemizygosity of the ATIII locus. In the remaining 15 kindreds, two copies of the ATIII gene are present and appear to be grossly normal at the level of whole genome Southern blotting, suggesting that small deletions, insertions or limited nucleotide substitution(s) in the antithrombin III gene, or "trans-acting" defects at other loci involved in the processing, modification, and secretion of biologically active ATIII are responsible for the observed anticoagulant disorders.

Protein inhibitors of crystal growth.

Nephrocalcin is a urinary glycopeptide that may be a physiological inhibitor of nephrolithiasis. Monomeric nephrocalcin purified from ethylenediaminetetracetic acid-treated urine is 14,000 daltons. Compositional analyses indicate that nephrocalcin is 10 per cent carbohydrate by weight and that 25 per cent of the amino acid residues are acidic (glutamic acid, aspartic acid and gamma-carboxyglutamic acid). Nephrocalcin binds reversibly to calcium oxalate crystals with a dissociation constant of about 0.5 microM. The high collapse pressure of nephrocalcin, 41.5 dynes per cm., measured for a monolayer at the air-water interface, suggests a highly organized structure in which hydrophilic and hydrophobic regions occupy separate regions on the surface of the inhibitor. Nephrocalcin contains the unusual amino acid, gamma-carboxyglutamic acid. Nephrocalcin isolated from urine of stone formers and from kidney stones does not contain gamma-carboxyglutamic acid and it has altered surface properties compared to normal nephrocalcin. The presence of the gamma-carboxyglutamic acid modification and the ability to form stable films with high collapse pressures may be important factors enabling nephrocalcin to prevent stone formation in vivo. The blood of cold water fishes contains antifreeze glycopeptides and/or peptides to prevent it from freezing. The structure of one such antifreeze peptide and its interactions with the crystal lattice of hexagonal ice are discussed as a model for how nephrocalcin might interact with calcium oxalate crystals and arrest their growth in urine.

Characterization of an unusual DNA length polymorphism 5' to the human antithrombin III gene.

Nucleotide sequence analysis revealed that a DNA length polymorphism 5' to the human antithrombin III gene is due to the presence of 32bp or 108bp nonhomologous nucleotide sequences (variable segments) 345bp upstream from the translation initiation codon. Sequences at the 3' borders of both variable segments can form intrastrand inverted repeat structures with sequences further downstream. An inverted repeat is also found immediately 5' to the site where the variable segments are located. Thus, cruciform structures may form flanking the variable segments of both alleles of this DNA length polymorphism. DNA secondary structure may be detected with single strand specific nucleases. S1 nuclease sensitive sites were mapped in recombinant plasmids containing the cloned alleles of the ATIII length polymorphism. The site most sensitive to S1 is located upstream from the variable segments in an AT-rich segment flanked by 6bp direct repeats. A region of lesser nuclease sensitivity was also observed in the AT-rich loops formed between the inverted repeats 5' to the variable segments.

Antithrombin III Utah: proline-407 to leucine mutation in a highly conserved region near the inhibitor reactive site.

A dysfunctional antithrombin III (ATIII) gene encoding a qualitatively and quantitatively abnormal anticoagulant molecule is responsible for hereditary thrombosis in a Utah kindred [Bock et al. (1985) Am. J. Hum. Genet. 37, 32-41]. Nucleotide sequencing of the entire protein-encoding portion of the cloned ATIII-Utah gene revealed a C to T transitional mutation which converts proline-407 to leucine. Proline-407 is located 14 amino acids C-terminal to the reactive site arginine of ATIII in a core region of the molecule that has been highly conserved during evolution of the serine protease inhibitor (serpin) gene family. The location of this proline in the crystal structure of the homologous serpin alpha 1-antitrypsin suggests that the leucine substitution in ATIII-Utah may interfere with correct folding of the mutant gene product, leading to its rapid turnover and the low antithrombin levels observed in patient plasmas. The Pro-407 to Leu mutation does not interfere with binding of antithrombin III to heparin. Patient antithrombin III, isolated by affinity chromatography on heparin-Sepharose, was reacted with purified thrombin. ATIII encoded by the patient's normal gene formed protease-inhibitor complexes with thrombin, whereas the product of the ATIII-Utah gene did not. The Pro-407 to Leu mutation destroys a restriction site for the enzyme StuI, permitting rapid diagnosis of affected members of the Utah kindred by Southern blotting of genomic DNA.

Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide.

Proteins secreted by prokaryotic cells are synthesized as precursors containing an amino-terminal extension sequence or signal peptide. Although these signal peptides share little primary sequence homology, recent studies suggest that they function via common pathways during the transport process and that a common element may reside in their secondary structural characteristics. We are investigating the role of an idealized hydrophobic sequence with high potential for alpha-helix formation in the Escherichia coli alkaline phosphatase signal peptide. Here, amino-acid substitutions were made using site-directed mutagenesis to produce a mutant signal sequence containing nine consecutive leucine residues in the hydrophobic core segment. Transport studies with this mutant precursor indicate that mature alkaline phosphatase is correctly targeted to the E. coli periplasm and that processing of the precursor to the mature form of the enzyme is extremely rapid. In contrast, processing is slowed when the mutant signal sequence is lengthened by the insertion of five additional leucine residues and one serine.

Intron structure of the human antithrombin III gene differs from that of other members of the serine protease inhibitor superfamily.

Antithrombin III (ATIII) plays an integral role in the coagulation system by inhibiting thrombin and several other activated clotting factors. Inherited deficiency of ATIII is quite common and can result in life-threatening thrombotic complications. In order to understand the basis of ATIII deficiency, we have isolated and characterized the normal human ATIII gene from a recombinant Charon 4A bacteriophage genomic library. The ATIII gene contains six exons and five introns distributed over approximately 19 kilobases of DNA. The positions of introns in the ATIII gene were compared with other members of the serine protease inhibitor family which share 17-31% amino acid homology. When aligned to achieve maximal protein homology, only one of the ATIII introns corresponded to the four introns of rat angiotensinogen or human alpha 1-antitrypsin. Similarly, only one ATIII intron was homologous to the seven introns of chicken ovalbumin. We present two testable models to explain the discrepancy in intron positions among members of the serine protease inhibitor superfamily of genes.

Homologs of eleven loci on human chromosome 11 assigned to two owl monkey chromosomes.

We localized 11 loci mapped to human chromosome 11 to two chromosomes, 4 and 19 of owl monkey karyotype VI (2n = 49/50), by the use of somatic cell hybrids. Furthermore, using in situ hybridization to chromosomes of two owl monkey karyotypes, the HSTF1 oncogene locus was precisely localized on homologs 19q (K-VI) and 2q (K-II). Comparative analysis of available gene-mapping data among human, mouse, and owl monkey chromosomes revealed a pattern of evolutionary change in a syntenic group on human chromosome 11. These structural changes could be explained as having derived from a pericentric inversion of human chromosome region 11cen----q13 and a translocation involving human region 11q22----qter during primate evolution.

Partial glycosylation of antithrombin III asparagine-135 is caused by the serine in the third position of its N-glycosylation consensus sequence and is responsible for production of the beta-antithrombin III isoform with enhanced heparin affinity.

Two antithrombin III (ATIII) isoforms occur naturally in human plasma. The alpha-ATIII isoform has four N-linked oligosaccharides attached to asparagines 96, 135, 155, and 192. The beta-ATIII isoform lacks carbohydrate on asparagine-135 (N135), which is near the heparin binding site, and binds heparin with higher affinity than does alpha-ATIII. Two isoforms are also produced when the normal human ATIII cDNA sequence is expressed in baculovirus-infected insect cells, and the recombinant beta' isoform similarly binds heparin with higher affinity than the recombinant alpha' isoform. Consensus sequences (CSs) of the ATIII N-glycosylation sites are N-X-S for 135 and N-X-T for 96, 155, and 192. On the basis of database and in vitro glycosylation studies suggesting that N-X-S CSs are utilized less efficiently than N-X-T CSs, we hypothesized that the beta-ATIII isoform might result from inefficient core glycosylation of the N135 N-X-S CS due to the presence of a serine, rather than a threonine, in the third position. ATIIIs with N-X-S, N-X-T, and N-X-A consensus sequences were expressed in baculovirus-infected insect cells. In contrast to the N-X-S sequence, which expressed a mixture of alpha' and beta' molecules, the N-X-T variant produced alpha' exclusively, while the N-X-A variant produced beta' exclusively. Thus, serine in the third position of the N135 CS is responsible for its "partial" glycosylation and leads to production of beta-ATIII.(ABSTRACT TRUNCATED AT 250 WORDS)

CpG mutations in the reactive site of human C1 inhibitor.

C1 inhibitor plays an important role in the regulation of vascular permeability through its ability to inactivate enzymes which release polypeptide kinins. Dysfunctional C1 inhibitor molecules are present in the plasma of affected members of the Da and Ri hereditary angioneurotic edema kindreds. We constructed genomic libraries from Da and Ri patient DNAs which had been cleaved with BclI to generate a fragment containing 21 kilobases of the C1 inhibitor locus. C1 inhibitor gene-containing recombinants originating from mutant Da and Ri alleles were differentiated from those derived from normal alleles by linkage analysis using the intragenic HgiAI restriction fragment length polymorphism. Nucleotide sequencing of the complete protein-coding regions of the mutant alleles identified two different mutations in a CpG dinucleotide corresponding to the first two bases of arginine codon 444. These single base mutations changed the identity of the functionally critical P1 reactive site residue from arginine to cysteine (Da) or histidine (Ri). The additional cysteine residue in C1 inhibitor Da suggests how it is covalently bound to albumin in plasma. The presence of CpG dinucleotides in the codons specifying the P1 arginines of C1 inhibitor and antithrombin III explains the high incidence of histidine and cysteine substitutions observed among dysfunctional mutants of these serine protease inhibitors.

The oligosaccharide side chain on Asn-135 of alpha-antithrombin, absent in beta-antithrombin, decreases the heparin affinity of the inhibitor by affecting the heparin-induced conformational change.

The beta-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated alpha-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of alpha- and beta-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for alpha-antithrombin than for beta-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrease in heparin binding affinity.

Human apolipoprotein B cDNA clone isolation and demonstration that liver apolipoprotein B mRNA is 22 kilobases in length.

An expression library made in plasmids pUC8 and pUC9 with mRNA derived from the human hepatoma cell line HepG2 was screened with a rabbit antiserum to human low density lipoprotein (LDL). Approximately 12,000 clones were screened and five positives were identified. The cDNA inserts were all 1500-1600 base pairs in length. The insert from one clone, pB8, was isolated, labeled by nicktranslation, and found to cross-hybridize strongly with the other four cDNA clones. The pB8 clone produces a fusion protein of approximately equal to 37.5 kDa that reacts in electrophoretic transfer blot analysis with rabbit anti-human LDL. This reactivity can be abolished by pretreatment of the antiserum with purified human LDL, p = 1.025 - 1.050 g/ml. A pB8-derived probe was used to demonstrate that apolipoprotein B (apo B) mRNA is present in HepG2 cells and liver extracts but not in HeLa cells or extracts from small intestine, heart, aorta, spleen, brain, skeletal muscle, lung, kidney, or ovary. RNA transfer blot analysis revealed that HepG2 cell apo B mRNA was approximately equal to 22 kilobases in length. These cDNA clones should allow the isolation of the apo B gene and ultimately the elucidation of the primary structure of this protein.

Identification of the antithrombin III heparin binding site.

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring beta-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Björk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-A long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central beta-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

Assignment of the human antithrombin III structural gene to chromosome 1q23-25.

The human antithrombin III (ATIII) structural gene was mapped by in situ hybridization and quantitative analysis of ATIII gene dosage in DNA isolated from carriers of chromosome 1 deletions. These studies indicate that the ATIII structural gene maps to human chromosome q23-q25 and so is likely identical to AT3.

Role of arginine 129 in heparin binding and activation of antithrombin.

The contribution of Arg(129) of the serpin, antithrombin, to the mechanism of allosteric activation of the protein by heparin was determined from the effect of mutating this residue to either His or Gln. R129H and R129Q antithrombins bound pentasaccharide and full-length heparins containing the antithrombin recognition sequence with similar large reductions in affinity ranging from 400- to 2500-fold relative to the control serpin, corresponding to a loss of 28-35% of the binding free energy. The salt dependence of pentasaccharide binding showed that the binding defect of the mutant serpin resulted from the loss of approximately 2 ionic interactions, suggesting that Arg(129) binds the pentasaccharide cooperatively with other residues. Rapid kinetic studies showed that the mutation minimally affected the initial low affinity binding of heparin to antithrombin, but greatly affected the subsequent conformational activation of the serpin leading to high affinity heparin binding, although not enough to disfavor activation. Consistent with these findings, the mutant antithrombin was normally activated by heparin for accelerated inhibition of factor Xa and thrombin. These results support an important role for Arg(129) in an induced-fit mechanism of heparin activation of antithrombin wherein conformational activation of the serpin positions Arg(129) and other residues for cooperative interactions with the heparin pentasaccharide so as to lock the serpin in the activated state.

Lysine 114 of antithrombin is of crucial importance for the affinity and kinetics of heparin pentasaccharide binding.

Lys(114) of the plasma coagulation proteinase inhibitor, antithrombin, has been implicated in binding of the glycosaminoglycan activator, heparin, by previous mutagenesis studies and by the crystal structure of antithrombin in complex with the active pentasaccharide unit of heparin. In the present work, substitution of Lys(114) by Ala or Met was shown to decrease the affinity of antithrombin for heparin and the pentasaccharide by approximately 10(5)-fold at I 0.15, corresponding to a reduction in binding energy of approximately 50%. The decrease in affinity was due to the loss of two to three ionic interactions, consistent with Lys(114) and at least one other basic residue of the inhibitor binding cooperatively to heparin, as well as to substantial nonionic interactions. The mutation minimally affected the initial, weak binding of the two-step mechanism of pentasaccharide binding to antithrombin but appreciably (>40-fold) decreased the forward rate constant of the conformational change in the second step and greatly (>1000-fold) increased the reverse rate constant of this step. Lys(114) is thus of greater importance for the affinity of heparin binding than any of the other antithrombin residues investigated so far, viz. Arg(47), Lys(125), and Arg(129). It contributes more than Arg(47) and Arg(129) to increasing the rate of induction of the activating conformational change, a role presumably exerted by interactions with the nonreducing end trisaccharide unit of the heparin pentasaccharide. However, its major effect, also larger than that of these two residues, is in maintaining antithrombin in the activated state by interactions that most likely involve the reducing end disaccharide unit.

A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase.

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.