Identification of the calmodulin-binding domain of recombinant calcium-independent phospholipase A2beta. implications for structure and function.

Calcium-independent phospholipase A(2) (iPLA(2)) is the major phospholipase A(2) activity in many cell types, and at least one isoform of this enzyme class is physically and functionally coupled to calmodulin (CaM) in a reversible calcium-dependent fashion. To identify the domain in recombinant iPLA(2)beta (riPLA(2)beta) underlying this interaction, multiple techniques were employed. First, we identified calcium-activated CaM induced alterations in the kinetics of proteolytic fragment generation during limited trypsinolysis (i.e. CaM footprinting). Tryptic digests of riPLA(2)beta (83 kDa) in the presence of EGTA alone, Ca(+2) alone, or EGTA and CaM together resulted in the production of a major 68-kDa protein whose kinetic rate of formation was specifically attenuated in incubations containing CaM and Ca(+2) together. Western blotting utilizing antibodies directed against either the N- or C-terminal regions of riPLA(2)beta indicated the specific protection of riPLA(2)beta by calcium-activated CaM at a cleavage site approximately 15 kDa from the C terminus. Moreover, calcium-activated calmodulin increased the kinetic rate of tryptic cleavage near the active site of riPLA(2)beta. Second, functional characterization of products from these partial tryptic digests demonstrated that approximately 90% of the 68-kDa riPLA(2)beta tryptic product (i.e. lacking the 15-kDa C-terminus) did not bind to a CaM affinity matrix in the presence of Ca(2+), although >95% of the noncleaved riPLA(2)beta as well as a 40-kDa C-terminal peptide bound tightly under these conditions. Third, when purified riPLA(2)beta was subjected to exhaustive trypsinolysis followed by ternary complex CaM affinity chromatography, a unique tryptic peptide ((694)AWSEMVGIQYFR(705)) within the 15-kDa C-terminal fragment was identified by RP-HPLC, which bound to CaM-agarose in the presence but not the absence of calcium ion. Fourth, fluorescence energy transfer experiments demonstrated that this peptide (694) bound to dansyl-calmodulin in a calcium-dependent fashion. Collectively, these results identify multiple contact points in the 15-kDa C terminus as being the major but not necessarily the only binding site responsible for the calcium-dependent regulation of iPLA(2)beta by CaM.

The genomic organization, complete mRNA sequence, cloning, and expression of a novel human intracellular membrane-associated calcium-independent phospholipase A(2).

During the sequencing of the long arm of chromosome 7 in the Human Genome Project, a predicted protein product of 40 kDa was identified, which contained two approximately 10-amino acid segments homologous to the ATP and lipase consensus sequences present in the founding members of a family of calcium-independent phospholipases A(2). Detailed inspection of the identified sequence (residues 79, 671-109,912 GenBank accession no. AC005058) demonstrated that it represented only a partial sequence of a larger undefined polypeptide product. Accordingly, we identified the complete genomic organization of this putative phospholipase A(2) through analyses of previously published expressed sequence tags, PCR of human heart cDNA, and 5'-rapid amplification of cDNA ends. Polymerase chain reaction and Northern blotting demonstrated a 3.4-kilobase message, which encoded a polypeptide with a maximum calculated molecular weight of 88476.9. This 3.4-kilobase message was present in multiple human parenchymal tissues including heart, skeletal muscle, placenta, brain, liver, and pancreas. Cloning and expression of the protein encoded by this message in Sf9 cells resulted in the production of two proteins of apparent molecular masses of 77 and 63 kDa as assessed by Western analyses utilizing immunoaffinity-purified antibody. Membranes from Sf9 cells expressing recombinant protein released fatty acid from sn-2-radiolabeled phosphatidylcholine and plasmenylcholine up to 10-fold more rapidly than controls. The initial rate of fatty acid release from the membrane fraction was 0. 3 nmol/mg.min. The recombinant protein was entirely calcium-independent, had a pH optimum of 8.0, was inhibited by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (IC(50) = 3 microM), and was predominantly present in the membrane-associated fraction. Collectively, these results describe the genomic organization, complete mRNA sequence, and sn-2-lipase activity of a novel intracellular calcium-independent membrane-associated phospholipase A(2).

Type 2M von Willebrand disease: F606I and I662F mutations in the glycoprotein Ib binding domain selectively impair ristocetin- but not botrocetin-mediated binding of von Willebrand factor to platelets.

von Willebrand disease (vWD) is a common, autosomally inherited, bleeding disorder caused by quantitative and/or qualitative deficiency of von Willebrand factor (vWF). We describe two families with a variant form of vWD where affected members of both families have borderline or low vWF antigen levels, normal vWF multimer patterns, disproportionately low ristocetin cofactor activity, and significant bleeding symptoms. Whereas ristocetin-induced binding of plasma vWF from affected members of both families to fixed platelets was reduced, botrocetin-induced platelet binding was normal. The sequencing of genomic DNA identified unique missense mutations in each family in the vWF exon 28. In Family A, a missense mutation at nucleotide 4105T --> A resulted in a Phe606Ile amino acid substitution (F606I) and in Family B, a missense mutation at nucleotide 4273A --> T resulted in an Ile662Phe amino acid substitution (I662F). Both mutations are within the large disulfide loop between Cys509 and Cys695 in the A1 domain that mediates vWF interaction with platelet glycoprotein Ib. Expression of recombinant vWF containing either F606I or I662F mutations resulted in mutant recombinant vWF with decreased ristocetin-induced platelet binding, but normal multimer structure, botrocetin-induced platelet binding, collagen binding, and binding to the conformation-sensitive monoclonal antibody, AvW-3. Both mutations are phenotypically distinct from the previously reported variant type 2MMilwaukee-1 because of the presence of normal botrocetin-induced platelet binding, collagen binding, and AvW-3 binding, as well as the greater frequency and intensity of clinical bleeding. When the reported type 2M mutations are mapped on the predicted three-dimensional structure of the A1 loop of vWF, the mutations cluster in one region that is distinct from the region in which the type 2B mutations cluster.

Type 2M:Milwaukee-1 von Willebrand disease: an in-frame deletion in the Cys509-Cys695 loop of the von Willebrand factor A1 domain causes deficient binding of von Willebrand factor to platelets.

This report examines the genetic basis of a variant form of moderately severe von Willebrand disease (vWD) characterized by low plasma von Willebrand factor antigen (vWF:Ag) levels and normal multimerization, typical of type 1 vWD, but disproportionately-low agonist-mediated platelet-binding activity. We identified an in-frame deletion in vWF exon 28 in three generations of affected family members, who are heterozygous for this mutation. The deletion of nucleotides 4,173-4,205 results in the loss of amino acids Arg629-Gln639 in the Cys509-Cys695 loop of the A1 domain in mature vWF. The secreted mutant vWF showed a normal multimeric profile but did not bind to platelets in the presence of optimal concentrations of either ristocetin or botrocetin. The mutant vWF also failed to interact with heparin, and with vWF monoclonal antibody AvW3, which blocks the binding of vWF to GPlb. In addition, mutant vWF showed reduced secretion from transfected cells concomitant with increased intracellular levels. These results confirm that the deletion is the genetic defect responsible for the reduced interaction of vWF with platelets. We have designated this new variant type 2M:Milwaukee-1 vWD. Our analysis suggests that the potential frequency of this phenotype in individuals diagnosed with type 1 vWD is about 0.5%.

Characterization of partial gene deletions in type III von Willebrand disease with alloantibody inhibitors.

von Willebrand factor gene deletions were characterized in four patients with severe type III von Wilebrand disease and alloantibodies to von Willebrand factor. A PCR-based strategy was used to characterize the boundaries of the deletions. Identical 30 kb von Willebrand factor gene deletions which include exons 33 through 38 were identified in two siblings of one family by this method. A small 5 base pair insertion (CCTGG) was sequenced at the deletion breakpoint. PCR analysis was used to detect the deletion in three generations of the family, including two family members who are heterozygous for the deletion. In a second family, two type III vWD patients, who are distant cousins, share an approximately 56 kb deletion of exons 22 through 43. The identification and characterization of large vWF gene deletions in these type III vWD patients provides further support for the association between large deletions in both von Willebrand factor alleles and the development of inhibitory alloantibodies.

von Willebrand disease type B: a missense mutation selectively abolishes ristocetin-induced von Willebrand factor binding to platelet glycoprotein Ib.

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.

Molecular basis of von Willebrand disease type IIB. Candidate mutations cluster in one disulfide loop between proposed platelet glycoprotein Ib binding sequences.

Many variants of von Willebrand disease (vWD) with qualitatively abnormal von Willebrand factor (vWF) are recognized. In vWD type IIB, the abnormal protein displays enhanced affinity for a platelet vWF receptor, the glycoprotein Ib-IX complex. 14 patients from 7 unrelated families with vWD type IIB were studied to determine the molecular basis for this phenotype. Specific oligonucleotide primers were used to amplify portions of vWF exon 28 encoding a domain that interacts with the platelet glycoprotein Ib-IX complex. Candidate missense mutations were identified for all 14 patients by DNA sequencing, allele specific oligonucleotide hybridization, and restriction endonuclease digestion. These sequence changes occur in an 11 amino acid segment within a single disulfide loop bounded by Cys(509) and Cys(695). All of these sequence changes are C----T transitions within CG dinucleotides. Six patients from two unrelated families were heterozygous for the encoded sequence Arg(543)----Trp. Seven patients from four unrelated families were heterozygous for the encoded sequence Arg(545)----Cys; this sequence change appears to have occurred independently three times, once as a new spontaneous mutation. One patient with apparently sporadic vWD type IIB was heterozygous for the encoded sequence Val(553)----Met, and this appears to be a new mutation. None of these sequence changes was found in 100 normal alleles. These findings suggest that vWD type IIB may be caused by relatively few distinct mutations, that these mutations may cluster within a specific region of one disulfide loop in vWF domain A1, and that this region can modulate the affinity of vWF for the platelet glycoprotein Ib-IX complex.

Human von Willebrand factor gene and pseudogene: structural analysis and differentiation by polymerase chain reaction.

Structural analysis of the von Willebrand factor gene located on chromosome 12 is complicated by the presence of a partial unprocessed pseudogene on chromosome 22q11-13. The structures of the von Willebrand factor pseudogene and corresponding segment of the gene were determined, and methods were developed for the rapid differentiation of von Willebrand factor gene and pseudogene sequences. The pseudogene is 21-29 kilobases in length and corresponds to 12 exons (exons 23-34) of the von Willebrand factor gene. Approximately 21 kilobases of the gene and pseudogene were sequenced, including the 5' boundary of the pseudogene. The 3' boundary of the pseudogene lies within an 8-kb region corresponding to intron 34 of the gene. The presence of splice site and nonsense mutations suggests that the pseudogene cannot yield functional transcripts. The pseudogene has diverged approximately 3.1% in nucleotide sequence from the gene. This suggests a recent evolutionary origin approximately 19-29 million years ago, near the time of divergence of humans and apes from monkeys. Several repetitive sequences were identified, including 4 Alu, one Line-1, and several short simple sequence repeats. Several of these simple repeats differ in length between the gene and pseudogene and provide useful markers for distinguishing these loci. Sequence differences between the gene and pseudogene were exploited to design oligonucleotide primers for use in the polymerase chain reaction to selectivity amplify sequences corresponding to exons 23-34 from either the von Willebrand factor gene or the pseudogene. This method is useful for the analysis of gene defects in patients with von Willebrand disease, without interference from homologous sequences in the pseudogene.

Hepatic extraction of organic anions in the rat depends on ligand hydrophobicity.

Non-bile-salt cholephilic organic anions are efficiently taken up by the liver. Recent work from our group has suggested the possible importance of relative hydrophobicity among various organic anions in hepatic uptake. To further validate and clarify this, we studied hepatic extraction of five different cholephilic dyes using the isolated perfused rat liver in single-pass mode. Albumin binding affinities and capacities for each of the ligands were measured in vitro to permit evaluation of in vivo interactions for each of them over a spectrum of unbound ligand concentrations. As expected, a strong positive correlation was found between ligand hydrophobicity and the relative degree of albumin binding affinity and capacity. Using appropriate experimental conditions, we also found a strong positive correlation between hepatic extraction efficiency for a given ligand and both its hydrophobicity and its unbound concentration. These data indicate that where the unbound ligand concentration is significant, the greater the ligand hydrophobicity, the greater is its efficiency of hepatic extraction. We conclude that hepatic extraction efficiency for non-bile-salt cholephilic organic anions depends on a combination of ligand hydrophilic/hydrophobic balance and the availability of the unbound ligand for uptake.

Severe type III von Willebrand's disease caused by deletion of exon 42 of the von Willebrand factor gene: family studies that identify carriers of the condition and a compound heterozygous individual.

Southern blotting was performed with cDNA probes for the human von Willebrand factor (vWF) gene on six patients with severe type III von Willebrand's disease (vWD). A partial deletion in the 3' end of the vWF gene was demonstrated in one individual whose parents were related and who had an alloantibody inhibitor to vWF. A resulting novel 2.0-kilobase (kb) EcoRI fragment was used for carrier detection within the patient's family, and seven carriers of this recessive trait were identified. Of the six tested, five had normal or only slightly reduced levels of vWF antigen, but with generally higher levels of factor VIII. The sixth carrier had moderately severe vWD and it is proposed that this patient is heterozygous for the defective vWF gene and a second recessive vWF defect. The novel 2.0-kb EcoRI restriction fragment was cloned and sequenced, and compared with that of the corresponding normal 4.2-kb EcoRI fragment that includes exons 41 and 42 of the vWF gene. A deletion of 2,320 base pairs (bp) which included exon 42, was identified and a novel 182-bp insert was found between the breakpoints. This insert was detected by polymerase chain reaction amplification both in the patient's DNA and in his carrier relatives.

Structure of the gene for human von Willebrand factor.

von Willebrand factor is a large multimeric plasma protein composed of identical subunits which contain four types of repeated domains. von Willebrand factor is essential for normal hemostasis, and deficiency of von Willebrand factor is the most common inherited bleeding disorder of man. Four human genomic DNA cosmid libraries and one bacteriophage lambda library were screened with von Willebrand factor cDNA probes. Twenty positive overlapping clones were characterized that span the entire von Willebrand factor gene. A high-resolution restriction map was constructed for approximately 75% of the locus and a total of approximately 33.8 kilobases was sequenced on both strands including all intron-exon boundaries. The gene is approximately 178 kilobases in length and contains 52 exons. The exons vary from 40 to 1379 base pairs in length, and the introns vary from 97 base pairs to approximately 19.9 kilobases in length. The signal peptide and propeptide (von Willebrand antigen II) of von Willebrand factor are encoded by 17 exons in approximately 80 kilobases of DNA while the mature subunit of von Willebrand factor and 3' noncoding region are encoded by 35 exons in the remaining approximately 100 kilobases of the gene. A number of repetitive sequences were identified including 14 Alu repeats and a approximately 670-base pair TCTA simple repeat in intron 40 that is polymorphic. Regions of the gene that encode homologous domains have similar structures, supporting a model for their origin by gene segment duplication.

Biosynthesis of glucosyl monophosphoryl undecaprenol and its role in lipoteichoic acid biosynthesis.

A glucophospholipid was detected in an incubation mixture containing UDP-glucose, MgCl2, ATP, and a particulate enzyme prepared from Streptococcus sanguis. The synthesis of this lipid was inhibited strongly by UDP and moderately by UMP. The molar ratio of glucose to phosphate in the purified lipid was found to be 1:1. Glucose and glucose 1-phosphate were released by mild alkaline hydrolysis of the glucophospholipid. The lipid produced by mild acid degradation of the purified lipid yielded a thin-layer chromatographic profile similar to that of acid-treated undecaprenol. One of the minor components exhibited the same mobility as untreated undecaprenol. To characterize further the lipid moiety of the glucophospholipid, a polyisoprenol was purified from the neutral lipid of S. sanguis. The polyisoprenol was converted in the presence of ATP, UDP-glucose, and the particulate enzyme into a lipid which exhibited the same thin-layer chromatographic mobility as the glucophospholipid. The structure of the polyisoprenol was determined by nuclear magnetic resonance and mass spectrometry to be an undecaprenol with an internal cis-trans ratio of 7:2. These results indicate that the glucophospholipid is glucosyl monophosphoryl undecaprenol. The glucosyl moiety of the glucophospholipid was shown to be incorporated in the presence of the particulate enzyme into a macromolecule which was characterized as a lipoteichoic acid by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and DEAE-cellulose column chromatography. This result indicates that glucosyl monophosphoryl undecaprenol is the direct glucosyl donor in the synthesis of lipoteichoic acid.

Analytical gradient polyacrylamide gel-crossed line immunoelectrophores. A technique for locating species specific Mycobacterium tuberculosis H37RV antigens in polyacrylamide gel columns.

Analytical gradient polyacrylamide gel electrophoresis of antigens from Mycobacterium tuberculosis H37RV was combined with crossed line immunoelectrophoresis to locate the position of antigens in polyacrylamide gel columns that appeared to be specific for M. tuberculosis H37RV. Specificity was established by comparing H37RV with M. kansaii CE in the electrophoretic procedure and the relative mobility (Rm) values are being used in an effort to recover and enrich specific antigens from preparative gel columns. Such fractions should be more suitable in efforts to recover purified monospecific components from M. tuberculosis H37RV.

Biosynthesis of glycosylated glycerolphosphate polymers in Streptococcus sanguis.

Two types of glycosylated glycerolphosphates were synthesized when a particulate enzyme prepared from Streptococcus sanguis was incubated with [3H]-phosphatidylglycerol and uridine diphosphate-[14C]glucose in the presence of MgCl2. The first type was extractable with saline and contained no fatty acid. The second type was pellet bound and could be extracted with 0.1% sodium dodecyl sulfate. Both types of polymers were purified and partially characterized. The first type of polymer was fractionated into five polymers, peaks 2a, 2b, 2c, 3a, and 3b. All except peak 2a, which contained only [3H]glycerol, contained both [3H]glycerol and [14C]glucose. [3H]NaBH4 reduction of acid hydrolysates of the polymers revealed that all of the polymers contained glucose as the major sugar componenta nd xylose as the minor sugar component. The second type of polymer was fractionated into three polymers, P-1, P-2, and P-3. All contained [3H]-glycerol, [14C]glucose, and fatty acids. P-1 appeared to be pure, whereas P-2 and P-3 contained two polymers each, as judged from sodium dodecyl sulfate-polyacrylamide gel electrophoresis.