Localization by site-directed mutagenesis of the site in human complement factor H that binds to Streptococcus pyogenes M protein

M-protein receptors located on Streptococcus pyogenes cells are known to bind human plasma protein factor H. Human factor H is composed of 20 short consensus repeat (SCR) domains containing approximately 60 amino acids each. Factor H controls the activation of the alternative pathway of complement in plasma. We have scanned the entire human factor H molecule by site-directed deletion mutagenesis, expressed the recombinant proteins in insect cells using the baculovirus system, and measured the binding of different purified mutant proteins to three strains of S. pyogenes. These studies have revealed that recombinant factor H lacking SCR domains 6 to 10 does not bind to wild-type M+ S. pyogenes JRS4. Experiments performed with S. pyogenes JRS251, in which both C-repeat domains of M protein were deleted, demonstrated that all of the factor H mutant proteins bound weakly to these cells except those lacking the SCR region from domains 6 to 10. Neither human factor H nor any of the recombinant proteins bound to the M- strain JRS145. Our results indicate that the only binding site on human factor H that interacts with streptococcus M protein is located in SCR domains 6 to 10 of factor H and that regions of M protein outside the C-repeat domains are involved in binding factor H.     

Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus

Immunoblots using bovine antibody against Haemophilus somnus as the primary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants expressed proteins which were recognized by bovine antiserum in Western blots (immunoblots). Ten of the recombinants expressing a 31-kDa protein caused the lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteriae, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonorrhoeae. An amino acid analysis of the recombinant 31-kDa protein agreed with the amino acid composition deduced from the DNA sequence.     

Identification of the region of alpha-catenin that plays an essential role in cadherin-mediated cell adhesion

alpha-Catenin is an intrinsic component of the cadherin adhesion complex and is a 102-kDa protein with multiple interaction sites, including homodimerization sites, and binding sites for beta- and gamma-catenin (plakoglobin), alpha-actinin, and actin. Besides the binding to beta- or gamma-catenin, it is unknown, however, which interaction is critical for the function of cadherins. By expressing a series of E-cadherin-alpha-catenin chimeric molecules on leukemia cells (K562), we have identified the region of alpha-catenin that confers aggregation inducing activity to nonfunctional tail-less E-cadherin. The region has been mapped to the carboxyl-terminal 295 amino acids of alpha-catenin. Consistent with this result, expression in alpha-catenin-deficient cells (DLD-1/Delta alpha) of a mutant alpha-catenin molecule consisting of the amino-terminal beta-/gamma-catenin-binding site and the carboxyl-terminal cell adhesion region identified in the above experiments induced E-cadherin-mediated cell aggregation and compaction. Cells expressing E-cadherin chimeric molecules with the homologous carboxyl-terminal region of vinculin, which contains the actin-binding site of vinculin, did not, however, aggregate as strongly as ones expressing E-cadherin-alpha-catenin chimeric molecules.     

Assignment of the binding site for tissue plasminogen activator on human fibronectin

The tissue-type plasminogen activator (t-PA) has been found to bind reversibly to human fibronectin (Fn). To locate the binding site on Fn for t-PA, the Fn was degraded with N-tosyl-L-phenylalanyl chloromethyl ketone-treated trypsin, and the resulting fragments were monitored by the enzyme-linked immunosorbent assay method for t-PA binding activities. A 20-kDa fragment with t-PA binding activity was identified, separated, and purified. It was subjected to further degradation with Staphylococcus aureus proteinase V8. An active 10-kDa fragment was finally purified by reverse-phase high pressure liquid chromatography on a C3 column. The dissociation constants of the binding of Fn and the 10-kDa fragment to t-PA were estimated by Scatchard plot to be 1.13 x 10(-8) and 2.08 x 10(-8) M, respectively. The 10-kDa fragment was sequenced and proved to be located at the 8-9th domains of type I homology of Fn. Based on the structural analysis of the 8-9th domains, a heptadecapeptide corresponding to the sequence Thr535-Glyl551 of Fn, which resided at the large disulfide loop of domain (I-9), was designed and synthesized. Both the 10-kDa fragment and the synthetic peptide could competitively inhibit the binding of Fn to t-PA. The synthetic peptide showed about one-tenth of the binding activity of Fn to t-PA with a dissociation constant of 1.35 x 10(-7) M and was proved to be the binding region of Fn for t-PA. In addition, like the intact Fn, both the 10-kDa fragment and the synthetic peptide could remarkably enhance the amidolytic activity of t-PA in a dose-dependent manner, as shown by using S-2288 as a chromogenic substrate.     

Direct binding of p130(Cas) to the guanine nucleotide exchange factor C3G

p130(Cas) (Cas; crk-associated substrate) belongs to a new family of docking molecules. It contains one Src homology (SH) 3 domain in its amino-terminal region followed by a region containing binding motifs for SH2 and SH3 domains. To gain further insight into Cas signaling we used the SH3 domain of Cas in a two-hybrid screen to search a human placenta library for binding partners. The screen confirmed a previous finding of its binding to the focal adhesion kinase (FAK) but also identified C3G, a guanine nucleotide exchange factor. We found direct interaction between Cas and C3G in vitro and in vivo. A series of analysis with C3G deletion mutants revealed a proline-rich Cas-binding site (Ala0-Pro1-Pro2-Lys3-Pro4-Pro5-Leu6-Pro7) located NH2-terminal to the previously characterized Crk binding motifs in C3G. Mutagenesis studies showed that Pro1, Lys3, and Pro4 within the ligand-binding site are critical for high affinity interaction. These results, combined with sequence alignments of proline-rich binding elements from proteins known for Cas binding, define the consensus sequence XXPXKPX which is recognized by the CasSH3 domain. Cas shows structural characteristics of a docking molecule and may serve to bring C3G to specific compartments within the cell.     

Proteolysis of Saccharomyces cerevisiae RNase H1 in E coli

Expression of S cerevisiae RNase H1 in E coli leads to the formation of a proteolytic product with a molecular mass of 30 kDa that is derived from the 39-kDa full length protein. The 30-kDa form retains RNase H1 activity, as determined by renaturation gel assay. The amount of proteolysis observed depends on the procedure used in preparing the cell extracts for protein analysis. The cleavage site on the amino acid sequence of the 39-kDa RNase H1 was determined by N-terminal sequence analysis of the 30-kDa proteolytic form. The cut occurs between two arginines located at the amino terminus region of the protein. The pattern of proteolysis was examined for both the wild-type RNase H1 and a mutant RNase H1 that was constructed in this work. In the mutant the second arginine of the cleavage site was changed to a lysine. Comparisons of the expression of the wild-type and altered protein in two different E coli strains demonstrate that the protease responsible for the degradation has a specificity very similar to that of the OmpT protease. However, the proteolysis observed in an OmpT background in extracts, prepared by boiling the cells in SDS containing buffer, indicates that the protease may, unlike OH108.     

Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA

Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. Atkinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol. 15:233-241, 1996) to protect mice against lethal infection by this organism. In this study, colony blot analysis of an A. hydrophila genomic library using antiserum to A. hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads. The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli. ORF1 was subsequently designated ilpA (immunophilin-like protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E. coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila. ORF3 was designated fkpA (FK506 binding protein) by analogy with the E. coli FkpA protein. Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA. hydrophila 30-kDa membrane protein. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in the virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.     

Identification of a 71-kilodalton surface-associated Hsp70 homologue in Coxiella burnetii

A Coxiella burnetii Hsp70 homologue was identified by using an acid activation in vitro system in which protein synthesis has been followed by [35S]methionine labeling, autoradiography, and immunoblotting. The protein was one of those predominantly labeled, and the immunoblots revealed that it was recognized by anti-DnaK antibodies. The corresponding gene was isolated, and its nucleotide sequence was determined and analyzed. A single open reading frame (ORF) with a size of 1,968 bp was identified. The ORF encodes a protein containing 656 residues and having a molecular weight of 70, 800. The -10 promoter sequence was shown to be identical with the consensus heat shock sigma32 promoter sequence. The base composition at the presumed -35 region revealed an EcoRI site in the expected region, which is assumed to be located at the border of the cloned fragment. The gene was expressed in Escherichia coli as an intact protein. The C. burnetii 71-kDa protein sequence has a high degree of homology to sequences of the Hsp70 family. A comparison of sequences revealed that the similarity with Hsp70s from other intracellular bacteria, e.g., Legionella pneumophila and Francisella tularensis, as well as E. coli DnaK, is more than 80%. The homologous regions are found in the N-terminal and central parts of the protein sequence, and they include the signature patterns of the Hsp70 family of proteins. The presence of the 71-kDa protein in association with the cell wall as well as in the cytoplasm was demonstrated by the use of immunoelectron microscopy. The dual localization was verified by Western blot analysis of proteins in C. burnetii cell fractions, using purified antibodies directed to the 71-kDa protein.     

Identification of benz(othi)azepine-binding regions within L-type calcium channel alpha1 subunits

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel alpha1 subunits we synthesized the benzazepine [3H]benziazem as a novel photoaffinity probe. [3H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca2+ channels with high affinity (Kd = 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [3H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca2+ antagonists, must be located in close proximity to pore-forming regions of the channel.     

Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase

Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Arg1487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Arg-Gly-Ile-Tyr-Arg1500 , located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of cAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type II regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-(1189-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(1189-1525). Binding studies with PKA and the non-hydrolysable ATP analogue [14C]fluorosulfonylbenzoyladenosine and [3H]cAMP do not reveal any influence of the short HCV-derived peptides or HCV-polyprotein-(1189-1525) upon the affinity of PKA for these nucleotides. The complex interactions of the NS3 fragments could influence one of the most important signal pathways of the cell and, therefore, could possibly provide new pathological mechanisms for HCV infections of liver.     

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.     

Characterization of the hypoxia-inducible protein binding site within the pyrimidine-rich tract in the 3'-untranslated region of the tyrosine hydroxylase mRNA

Reduced tension of O2 slows the degradation rate of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. The observed increase in half-life (30 h versus 10 h) correlates with enhanced binding of a 66-kDa protein (hypoxia inducible protein) to the pyrimidine-rich tract located between bases 1552 1578 in the 3 -untranslated region of TH mRNA (hypoxia-inducible protein binding site (HIPBS)). The present study investigates the protein binding site within the 27-base HIPBS, first by using specific cleavages of HIPBS and its flanking sequences with antisense oligodeoxynucleotides and RNase H and then by using mutational analysis of the binding properties. We found that the 27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia-stimulated manner. We further identified the optimal hypoxia-inducible protein binding site that is represented by the motif (U/C)(C/U)CCCU, where the core binding site is indicated by the underlined cytidines. Substitutions of either one of the cytidines with purine or uridine abolished the protein binding. The mutations within HIPBS, which partially reduced binding, did not prevent stimulation of protein binding for extracts from hypoxic cells. The hypoxia-induced increase in complex formation was proportional to the strength of binding using proteins from normoxic cells. The HIPBS element is conserved in TH mRNAs derived from different species.     

Nucleotide binding to the C-terminal nucleotide binding domain of ArsA. Studies with an ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine

ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment was not protected upon incubation with ATP alone, indicating that A2 might be unoccupied. However, upon incubation with ATP and antimonite, almost complete protection from trypsin was seen. ATP and FSBA together mimicked the effect of ATP and antimonite, implying that this fully protected conformation might be the result of both sites occupied with the nucleotide. It is proposed that the A1 site in ArsA is a high affinity ATP site, whereas the allosteric ligand antimonite is required to allow ATP binding to A2, resulting in catalytic cooperativity. Thus antimonite binding may act as a switch in regulating ATP binding to A2 and hence the ATPase activity of ArsA.     

Degradation of nonionic surfactants and polychlorinated biphenyls by recombinant field application vectors

Immunoglobin binding protein (BiP) molecules exist as both monomers and oligomers and phosphorylated BiP is restricted to the oligomeric pool. Modified BiP is not bound to proteins such as immunoglobulin heavy chain and consequently, may constitute an inactive form. Unlike earlier analysis of mammalian BiP isolated by two-dimensional gel electrophoresis, results here demonstrated that immunoprecipitated BiP displayed predominantly threonine phosphorylation with only a trace of detectable phosphoserine. Like other Hsp70 family members, BiP is comprised of three domains: an amino terminal domain which binds nucleotide, an 18 kilodalton domain which binds peptide, and a carboxyl terminal variable domain of unknown function. Cyanogen bromide cleavage and enzymatic digestion experiments mapped threonine phosphorylation to a site within a 47 amino acid sequence of the peptide binding domain which contains seven threonine residues. Partial proteinase K digestion in the presence of ATP independently verified that the in vivo phosphorylation site of mammalian (BiP) is located within the peptide binding domain. Furthermore, phosphorylation did not impede BiPs ATP-induced conformational change. Thus, the peptide binding domain of BiP is phosphorylated on threonine residue(s) mapping to not more than two tryptic fragments within the peptide binding domain. This location on the molecule could explain why phosphorylated BiP is not detected bound to proteins in vivo.     

Zipper protein, a B-G protein with the ability to regulate actin/myosin 1 interactions in the intestinal brush border

We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and alpha helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush-border extracts. Using 5'-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin.     

Ultrastructural and biochemical properties of the 120-kDa form of chick kinectin

Kinectin, an integral membrane protein (160 kDa), was identified as a kinesin-binding protein. Analysis of the predicted amino acid sequence of kinectin cDNA indicated an alpha-helical coiled-coil structure from amino acid 320 to 1310. A 120-kDa kinectin has been observed consistently, and N-terminal sequencing showed that 232 amino acids were missing from the N terminus of full-length kinectin. 120-kDa kinectin was distributed in the supernatant and a low density fraction of vesicles, whereas both forms were in the high density fraction of vesicles. In the electron microscope, the 120-kDa form appeared as a linear molecule of 133 nm in length. In hydrodynamic studies, the cytosolic 120-kDa kinectin was a dimer. Monoclonal antibody molecules (anti-kinectin KR160.9) bound asymmetrically to kinectin often with two antibodies/kinectin, indicative of a parallel coiled-coil. Metabolic labeling with [3H]myristic acid showed that both the 120- and 160-kDa kinectin are myristoylated in chick embryo fibroblasts. The myristoylation of 120-kDa kinectin may provide a mechanism for linking it to a low density fraction of vesicles. Immunoprecipitation with a 160-kDa kinectin-specific antibody brought down the 120-kDa kinectin. Thus, we suggest that kinectin is an extended parallel coiled-coil dimer, often a heterodimer.