Prediction of the membrane-spanning beta-strands of the major outer membrane protein of Chlamydia

There is preliminary experimental evidence indicating that the major outer-membrane protein (MOMP) of Chlamydia is a porin. We tested this hypothesis for the MOMP of the mouse pneumonitis serovar of Chlamydia trachomatis using two secondary structure prediction methods. First, an algorithm that calculates the mean hydrophobicity of one side of putative beta-strands predicted the positions of 16 transmembrane segments, a structure common to known porins. Second, outer loops typical of porins were assigned using an artificial neural network trained to predict the topology of bacterial outer-membrane proteins with a predominance of beta-strands. A topology model based on these results locates the four variable domains (VDs) of the MOMP on the outer loops and the five constant domains on beta-strands and the periplasmic turns. This model is consistent with genetic analysis and immunological and biochemical data that indicate the VDs are surface exposed. Furthermore, it shows significant homology with the consensus porin model of the program FORESST, which contrasts a proposed secondary structure against a data set of 349 proteins of known structure. Analysis of the MOMP of other chlamydial species corroborated our predicted model.     

Epitope mapping with solid-phase peptides: identification of type-, subspecies-, species- and genus-reactive antibody binding domains on the major outer membrane protein of Chlamydia trachomatis

The major outer membrane protein (MOMP) of Chlamydia trachomatis carries serovar-, subspecies-, species- and genus immunodomains, antibodies to which may be protective. We have compared the inferred amino acid sequences for MOMP from different serovars of C. trachomatis and from Chlamydia psittaci to identify the likely locations of these sero-taxonomic epitopes. Overlapping peptides corresponding to each of these regions were synthesized on a solid phase and probed with monoclonal antibodies (MAbs) of appropriate specificities. We describe the primary structures of the binding sites of MAb to each of these four epitopes on C. trachomatis serovar L1 MOMP.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

The major outer-membrane proteins of Chlamydia trachomatis serovars A and B: intra-serovar amino acid changes do not alter specificities of serovar- and C subspecies-reactive antibody-binding domains.

The major outer-membrane protein (MOMP) of Chlamydia trachomatis is a promising candidate antigen for chlamydial vaccine development. We have sequenced the MOMP genes for a serovar A and a serovar B isolate and have compared these new sequences with those already reported. Intra-serovar changes in the inferred amino acid sequences of the surface-exposed variable segments known to be responsible for binding of neutralizing antibody were observed. Nevertheless, epitope mapping with solid-phase peptides showed that these intra-serovar changes did not affect the binding of serovar- and subspecies-specific, potentially protective antibodies. Variable segment 1 of C. trachomatis serovar A contained two adjacent antibody-binding sites, one of which was C-subspecies specific while the other was serovar A specific. Therefore the subspecies binding site for C-complex organisms is in variable segment 1, whilst that for B-complex organisms is in variable segment 4. This work shows that MOMP sequences are relatively stable within the serovar categorization for isolates taken decades apart from different continents. Within a given serovar, however, limited interchange of functionally related amino acids may occur without impairing the binding of serovar-specific antibody.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Thiol peptides from the aspartate transaminase of chicken heart cytosol

Aspartate transaminase from chicken heart cytosol was immobilized covalently on activated thiol-Sepharose and digested with trypsin. After washing, the thiol-containing peptides were eluted with 2-mercaptoethanol and further purified by gel-filtration and paper chromatography. Three pure cysteinyl peptides were isolated. One of them may be represented as Ile-(Asp, Met, Cys, Gly, Leu, Thr2)-Lys; this peptide is identical to the fragment comprizing residues 387--395 in the peptide chain of aspartate transaminase from pig heart cytosol. It thus contains a cysteine residue homologous to Cys-390 of the pig heart enzyme. The second cysteinyl peptide had the following composition and partial sequence: Tyr-Phe-Val-Ser-Glu-Gly-Phe-Glu-Leu-Phe (Cys, Ala, Glu, Ser2, Phe)Lys, which corresponds to the sequence 242--258 of the pig enzyme and thus contains a cysteine residue homologous to Cys-252. The third cysteinyl peptide was similar to the tryptic peptide of the pig enzyme containing Cys-191.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Surface-exposed epitopes on the major outer-membrane protein of Chlamydia trachomatis defined with peptide antisera

The surface exposure of computer-predicted, linear B-cell epitopes on the major outer-membrane protein (MOMP) of Chlamydia trachomatis serovar B was assessed using antibodies raised against synthetic peptides in conjunction with immunogold transmission electron microscopy. Several of the chosen peptides elicited antibodies which reacted with both denatured and native MOMP. The majority of the exposed epitopes were found within the variable segments of MOMP. For each of the epitopes identified, the extent of their surface accessibility varied both among individual organisms and different developmental forms. Evidence for two distinct subspecies-specific epitopes within VS4 is presented.     

Evidence that Highly Conserved Residues of <Emphasis Type="Italic">Delonix regia</Emphasis> Trypsin Inhibitor Are Important for Activity

Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44–Cys89 and Cys139–Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4–5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139–Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44–Cys89) is not required.     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Engineering subtilisin E for enhanced stability and activity in polar organic solvents

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).     

A single peptide from the major outer membrane protein of Chlamydia trachomatis elicits T cell help for the production of antibodies to protective determinants.

The protective immune response to infection with Chlamydia trachomatis is associated with antibody reactivity to serovar-specific determinants on the major outer membrane protein (MOMP). Because this immunity is T cell dependent, it is essential to define those Th cell determinants that promote natural boosting of the protective antibody response. The gene for MOMP of serovar B was separated into nine overlapping fragments that represent the five C and four V regions. These fragments were expressed as fusion peptides with GST and used to identify the regions of the MOMP that contain T cell determinants recognized in BALB/c mice. We identified peptides that elicit a T cell response to Chlamydia by immunizing mice with the fusion peptides and testing the proliferative response of T cells in vitro to intact organism. For analysis of determinants seen after infection, animals were inoculated with live organism and the T cell proliferative response to each fusion peptide was measured in vitro. In contrast to proliferative analysis in which several regions of the MOMP elicited T cell responses, functional analysis demonstrated that a single fusion peptide, containing V segment three, elicited T cell help in vivo for the production of high titered antisera, specific for protective determinants on the MOMP.     

Perturbing the polar environment of Asp102 in trypsin: consequences of replacing conserved Ser214.

Much of the catalytic power of trypsin is derived from the unusual buried, charged side chain of Asp102. A polar cave provides the stabilization for maintaining the buried charge, and it features the conserved amino acid Ser214 adjacent to Asp102. Ser214 has been replaced with Ala, Glu, and Lys in rat anionic trypsin, and the consequences of these changes have been determined. Three-dimensional structures of the Glu and Lys variant trypsins reveal that the new 214 side chains are buried. The 2.2-A crystal structure (R = 0.150) of trypsin S214K shows that Lys214 occupies the position held by Ser214 and a buried water molecule in the buried polar cave. Lys214-N zeta is solvent inaccessible and is less than 5 A from the catalytic Asp102. The side chain of Glu214 (2.8 A, R = 0.168) in trypsin S214E shows two conformations. In the major one, the Glu carboxylate in S214E forms a hydrogen bond with Asp102. Analytical isoelectrofocusing results show that trypsin S214K has a significantly different isoelectric point than trypsin, corresponding to an additional positive charge. The kinetic parameter kcat demonstrates that, compared to trypsin, S214K has 1% of the catalytic activity on a tripeptide amide substrate and S214E is 44% as active. Electrostatic potential calculations provide corroboration of the charge on Lys214 and are consistent with the kinetic results, suggesting that the presence of Lys214 has disturbed the electrostatic potential of Asp102.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Amino acid sequences of two tryptic peptides from D(-)-beta-hydroxybutyrate dehydrogenase radiolabeled at essential carboxyl and sulfhydryl groups

D(-)beta-hydroxybutyrate dehydrogenase (BDH) purified from bovine heart mitochondria contains essential thiol and carboxyl groups. A tryptic BDH peptide labeled at an essential thiol with [3H]N-ethylmaleimide (NEM), and another tryptic peptide labeled at an essential carboxyl with N,N'-dicyclohexyl [14C]carbodiimide (DCCD), were isolated and sequenced. The peptide labeled with [3H]NEM had the sequence Met.Glu.Ser.Tyr.Cys*.Thr.Ser. Gly.Ser.Thr.Asp.Thr.Ser.Pro.Val.Ile.Lys. The label was at Cys. The same peptide was isolated from tryptic digests of BDH labeled at its nucleotide-binding site with the photoaffinity labeling reagent, arylazido- -[3-3H] alanyl-NAD. These results suggest that the essential thiol of BDH is located at its nucleotide-binding site, and agree with our previous observation that NAD and NADH protect BDH against inhibition by thiol modifiers. The [14C]DCCD-labeled peptide had the sequence Glu.Val.Ala.Glu*.Val. Asn. Leu.Trp.Gly.Thr.Val.Arg. DCCD appeared to modify the glutamic acid residue marked by an asterisk. Sequence analogies between these peptides and other proteins have been discussed.     

Characterization of Chlamydia trachomatis antigens with monoclonal and polyclonal antibodies

We used monoclonal antibodies (MAbs) to examine the antigenic specificity and biologic function of several Chlamydia trachomatis antigens. Thirteen distinct MAbs to eight C. trachomatis antigens were produced. Six MAbs reacted with unique epitopes on the major outer membrane protein (MOMP) and two of these had neutralizing activity. MAbs were produced to each of the chlamydial antigens with molecular masses of 10, 29, 32, 57, 60, 70, and 75 kilodaltons (kDa). These MAbs showed species and genus specificity in an immunoblot assay. None of the MAbs had neutralizing activity. The epitopes recognized on MOMP, 29-, and 10-kDa (presumably lipopolysaccharide) antigens were surface exposed. MAbs to the 75-kDa, 57-kDa, and MOMP antigens were used for immunoaffinity purification of these antigens to produce monospecific antisera in mice. With polyclonal sera, we found that the 75-kDa antigen was also immunoaccessible and that antibody to MOMP and 75-kDa antigens neutralized C. trachomatis infectivity. We conclude that, in addition to MOMP and lipopolysaccharide, antigens with molecular masses of 75 and 29 kDa are surface exposed. Antibodies to MOMP and 75-kDa antigens can neutralize the organism in vitro.     

Characterization of the disulfide bonds and free cysteine residues of the Chlamydia trachomatis mouse pneumonitis major outer membrane protein

Members of the genus Chlamydia lack a peptidoglycan layer. As a substitute for peptidoglycan, it has been proposed that several cysteine rich proteins, including the major outer membrane protein (MOMP), form disulfide bonds to provide rigidity to the cell wall. Alignment of the amino acids sequences of the MOMP from various serovars of Chlamydia showed that they have from 7 to 10 cysteine residues and seven of them are highly conserved. Which of these are free cysteine residues and which are involved in disulfide bonds is unknown. The complexity of the outer membrane of Chlamydia precludes at this point the characterization of the structure of the cysteines directly in the bacteria. Therefore, mass spectrometric analysis of a purified and refolded MOMP was used in this study. Characterization of the structure of this preparation of the MOMP is critical because it has been shown, in an animal model, to be a very effective vaccine against respiratory and genital infections. Here, we demonstrated that in this MOMP preparation four cysteines are involved in disulfide bonds, with intramolecular pairs formed between Cys(48) and Cys(55) and between Cys(201) and Cys(203). A stepwise alkylation, reduction, alkylation process using two different alkylating reagents was required to establish the Cys(48)-Cys(55) disulfide pair. The other residues in MOMP, Cys(51), Cys(136), Cys(226), and Cys(351), are free cysteines and could potentially form disulfide-linked complexes with other MOMP or other membrane proteins.     

Induction of HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein of Chlamydia trachomatis in human genital tract infections.

HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein (MOMP) of Chlamydia trachomatis are present in the peripheral blood of humans who acquired genital tract infections with the organism. Three HLA-A2-restricted epitopes and two HLA-B51-restricted epitopes were identified in serovar E-MOMP. One of the five epitopes spans a variable segment of MOMP and is likely a serovar E-specific epitope. The other four epitopes are localized in constant segments and are C. trachomatis species specific. CTL populations specific for one or more of the four constant segment epitopes were isolated from all 10 infected subjects tested, regardless of infecting serovars, but from only one of seven uninfected subjects tested. The CTLs failed to recognize corresponding peptides derived from Chlamydia pneumoniae MOMP, further suggesting that they indeed resulted from genital tract infections with C. trachomatis. Significantly, ME180 human cervical epithelial cells productively infected with C. trachomatis were killed by the MOMP peptide-specific CTLs. Further investigations of the ability of such CTLs to lyse normal infected epithelial cells and their presence at inflamed sites in the genital tract will help understand the protective or pathological role of CTLs in chlamydial infections. The MOMP CTL epitopes may be explored as potential components of a subunit vaccine against sexually transmitted diseases caused by C. trachomatis. Moreover, the knowledge provided here will facilitate studies of HLA class I pathways of chlamydial Ag processing and presentation in physiologically relevant human APCs.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage.