Sheath Protein Research Articles

Polypeptides of Treponema pallidum: progress toward understanding their structural, functional, and immunologic roles. Treponema Pallidum Polypeptide Research Group.

Treponema pallidum subsp. pallidum, the spirochete that causes syphilis, is unusual in a number of respects, including its small genome size, inability to grow under standard in vitro culture conditions, microaerophilism, apparent paucity of outer membrane proteins, structurally complex periplasmic flagella, and ability to evade the host immune responses and cause disease over a period of years to decades. Many of these attributes are related ultimately to its protein content. Our knowledge of the activities, structure, and immunogenicity of its proteins has been expanded by the application of recombinant DNA, hybridoma, and structural fractionation techniques. The purpose of this monograph is to summarize and correlate this new information by using two-dimensional gel electrophoresis, monoclonal antibody reactivity, sequence data, and other properties as the bases of polypeptide identification. The protein profiles of the T. pallidum subspecies causing syphilis, yaws, and endemic syphilis are virtually indistinguishable but differ considerably from those of other treponemal species. Among the most abundant polypeptides are a group of lipoproteins of unknown function that appear to be important in the immune response during syphilitic infection. The periplasmic flagella of T. pallidum and other spirochetes are unique with regard to their protein content and ultrastructure, as well as their periplasmic location. They are composed of three core proteins (homologous to the other members of the eubacterial flagellin family) and a single, unrelated sheath protein; the functional significance of this arrangement is not understood at present. Although the bacterium contains the chaperonins GroEL and DnaK, these proteins are not under the control of the heat shock regulon as they are in most organisms. Studies of the immunogenicity of T. pallidum proteins indicate that many may be useful for immunodiagnosis and immunoprotection. Future goals in T. pallidum polypeptide research include continued elucidation of their structural locations and functional activities, identification and characterization of the low-abundance outer membrane proteins, further study of the immunoprotective and immunodiagnostic potential of T. pallidum proteins, and clarification of the roles of treponemal proteins in pathogenesis.

Humoral immune response against Helicobacter pylori as determined by immunoblot.

An immunoblot method has been evaluated to diagnose Helicobacter pylori infection serologically by comparing 69 serum specimens from patients with a positive Gram stain and/or culture result and a positive urease test on biopsy material, as well as 51 serum specimens from patients with at least 4 negative urease tests, and negative microscopy and culture results. Sensitivity and specificity was found to be 100%. Recognition of the cross-reacting flagellin (66 kDa), flagellar sheath protein (51 kDa), and a 14 kDa protein are not a criterion for a current H. pylori infection. On the other hand, any combination of at least two of the 180, 120, 90, 75, 67, 29.5 and 19 kDa bands were diagnostic of infection. Three H. pylori strains, which were compared with both gel electrophoretic analyses and immunoblot reactivity, exhibited in part strong qualitative and quantitative differences that particularly affect the 120 kDa pathogenic factor, the large urease subunit and other proteins especially in the molecular mass range from 50 to 67 kDa. IgG immunoblot patterns showed that the choice of H. pylori strain, as well as a reproducible and standardizable antigen preparation, is of great importance for the reliability of serodiagnostic tests. The immunoblot method was found to be a valuable tool for the semi-quantitative confirmation of results achieved with other serological methods as well as optimization and quality control of the antigens used for serodiagnostic purposes.

Trans-splicing, of an early embryo mRNA in Litomosoides carinii, coding for the major microfilarial sheath protein gp22

Both genomic and cDNA clones have been isolated encoding the major sheath glycoprotein, gp22, of Litomosoides carinii microfilariae. The mature gp22 mRNA is shown to result from both trans-splicmg of a 22-nucleotide 5′-leader sequence to an acceptor site at position 313 of the pre-mRNA, immediately upstream from the start codon, and from cis-splicing of a 117-nt intron located within the coding sequence. Cis-splicing precedes the trans-splicing reaction. The gp22 reading frame of 148 codons has the inferred structure of a prepro-protein and includes a leader peptide and a pro-segment ahead of the known N terminus of the mature, extracellular protein of 105 amino acids. The N-terminal part of that protein contains five repeats of an elastin-related pentapeptide sequence, which, together with a proline-threonine segment between two Cys clusters in the center and at its C terminus, may cause an elongated conformation with an apparent molecular size of 22 kDa in contrast to the calculated M r of 11 200.

GTPase activity of bacteriophage T4 sheath protein

We show by nuclear magnetic resonance studies that, following GTP hydrolysis during phage T4 sheath contraction, GDP remains bound to the sheath protein (gp18), whereas orthophosphate is released. gp18 in the contracted state has GTPase activity and can hydrolyse exogenous GTP; the reaction is calcium-dependent and displays high substrate specificity. The process comprises two steps: (1) displacement of GDP from gp18 by exogenous GTP, and (2) GTP hydrolysis proper. The first step appears to be rate-limiting and to be accelerated when the nucleotide-protein interaction is mechanically disrupted by sonication.

Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95.

Human sperm tall fibrous sheath undergoes phosphorylation during its development

The phosphorylation of the human sperm tail fibrous sheath as a maturational step during its development is reported for the first time. This was demonstrated using GDA-J/F3 and RT97 monoclonal antibodies (MoAb) which recognize the fibrous sheath. In indirect immunofluorescence microscopy of frozen sections of human adult testes, the two antibodies reacted with the assembled fibrous sheath only, but the numbers of sperm tails stained with RT97 were consistently lower than those treated with GDA-J/F3. Furthermore, by using double indirect immunofluorescence, although the majority of spermatozoa were doubly stained with the two MoAbs, some GDA-J/F3-positive sperm tails were negative with RT97. In epididymal and ejaculated spermatozoa, the two antibodies stained all the tails. This indicated that the ontogenic appearance of the GDA-J/F3 epitope precedes that of RT97. In Western blotting and/or indirect immunofluorescence of spermatozoa, treatment of samples with alkaline phosphatase abolished the reactivity of RT97 while that of GDA-J/F3 MoAb was not affected. This finding indicated that the RT97 but not the GDA-J/F3 epitope was phosphorylated. Together, these results therefore reveal that during tail morphogenesis, the fibrous sheath undergoes phosphorylation as part of its structural maturation. Screening of sperm cell precursors recovered from oligozoospermic donors showed reaction of some abnormal germ cells with GDA-J/F3 MoAb but not with RT97, suggesting the possible failure of phosphorylation of the fibrous sheath protein in these cells. The significance of these findings is discussed together with the biological importance of phosphorylation to the fibrous sheath.

Localization of Sulfated Glycoprotein-2 (Clusterin) on Spermatozoa and in the Reproductive Tract of the Male Rat1

Sulfated glycoprotein-2 (SGP-2) is one of the major proteins secreted by rat Sertoli cells and epididymal cells in culture. The disulfide-linked dimeric protein secreted by Sertoli cells and found in seminiferous tubule fluid is composed of monomers of Mr 47 000 and 34 000 whereas the epididymal protein exhibits monomers of Mr 40 000 and 29 000. When both forms were chemically or enzymatically deglycosylated, they yielded proteins of similar molecular weight. No modification of the higher molecular weight testicular form by epididymal cells or fluids could be detected in incubation media. SGP-2 mRNA was localized in epididymal epithelium by in situ hybridization. Northern blot analysis indicated the testicular and epididymal mRNAs were of similar size. These findings suggest that the two forms of the protein occur because of tissue-specific post-translational modifications. The detergent-extracted protein from washed testicular spermatozoa is of the higher molecular weight form while epididymal sperm carry the lower molecular weight form. Immunohistochemical evidence suggests that the testicular form is removed prior to the initial segment of the epididymis and the epididymal form is applied in the proximal caput epididymidis. SGP-2 was immunolocalized to the sperm membrane at the ultrastructural level and was distinctly different from the immunolocalization of outer dense fiber proteins and fibrous sheath proteins.

Nucleotide sequence of the genes encoding the major tail sheath and tail tube proteins of bacteriophage P2

The major structural components of the contractile tail of bacteriophage P2 are proteins F 1 and F 11 which are believed to be the tail sheath and tube proteins, respectively. Both proteins were mapped previously to the P2 late gene F 1 based on the pattern of protein synthesis in various P2 amber mutants. In order to clarify the gene arrangement and to provide a basis for structural comparisons with other contractile phage tails, we have determined the nucleotide sequence of the region of the P2 genome encoding these two proteins. The coding regions were confirmed by location of the Fam4 mutation and by N-terminal amino acid sequencing of both proteins. The molecular weight and amino acid composition predicted by each of the coding regions correspond well to those determined experimentally for each protein. F 11 is encoded by a newly identified P2 late gene. These proteins bear little resemblance to their functional homologues in bacteriophage T4.

Expression of plasmid-encoded structural proteins permits engineering of bacteriophage T4 assembly

A complementation system for studying bacteriophage T4 tail assembly has been developed and used to test the effects of nonviable mutations on the function of a specific T4 tail protein, gp48. The complementation system assays the assembly function of gp48 without requiring that viable phage be produced, circumventing the operational problems of maintaining nonviable mutants of this lytic bacteriophage. The protein to be tested was preexpressed from cloned genes in a host cell prior to infection with the challenge phage. Assembly activity was assayed by monitoring the conversion of one tail assembly intermediate, the baseplate lacking gp48, into baseplates containing gp48 or into tube baseplates (or sheathed tails) assembled from such baseplates. Specific incorporation of gp48 into these structures was confirmed using gp48-specific antiserum, and the same serum was used in direct immunoelectron microscopy experiments to localize gp48 to the baseplate-proximal end of the T4 tail tube, at the site where the tube and sheath bind to the baseplate. The protein gp48 has been previously shown to be a baseplate protein, as well as a tail-tube-associated protein, and was tested for a possible role as a tail-length tape-measure protein. Tests with a deleted variant of gp48 were inconclusive because the protein was inactive. A variant of gp48, 20% longer than wild-type protein due to an internal duplication, was found to be partly functional in our assembly complementation system. This abnormally elongated protein allows several assembly steps to proceed, including the assembly of normal length T4 tails, implying that it does not specify tail length. The insertion-duplication variant of gp48 appears to have a defect in its interaction with the tail sheath protein, leading to abnormal sheath contraction.

Identification of a sheath-associated protein involved in phosphate transport in Sphaerotilus natans

Sphaerotilus natans was shown to have a fourfold lower K′mof phosphate transport when grown in medium containing 0.1 mm phosphate, compared to cells grown in 10.0 mm phosphate. Analysis of sheath proteins from cells grown at these two phosphate levels revealed a protein of 53 kDa present in the sheath of cells grown at a phosphate concentration of 0.1 mm. This sheath-associated, phosphate-regulated protein, designated SapP, was gel purified and used to raise a polyclonal antibody. Enzyme-linked immunosorbent assay was used to localize this protein to the surface of the sheathed cells. Phosphate uptake assays done in the presence of the antibody also showed a rise in the K′mof phosphate transport in cells grown in 0.1 mm phosphate, indicating that this protein is involved in high-affinity phosphate transport.

Expression in Escherichia coli of the 37-kilodalton endoflagellar sheath protein of Treponema pallidum by use of the polymerase chain reaction and a T7 expression system

We previously reported the complete primary structure of the 37-kilodalton endoflagellar sheath protein (FlaA) of Treponema pallidum. However, we were unable to determine the nucleotide sequence of flaA upstream of amino acid 10. The desired nucleotide sequence was obtained by use of a strategy based upon the polymerase chain reaction and was found to contain a consensus Escherichia coli promoter, a ribosomal binding site, and a 20-amino-acid signal peptide. Expression of FlaA in E. coli was achieved by cloning polymerase chain reaction-derived constructs lacking the native T. pallidum promoter into a temperature-inducible T7 expression system. Pulse-chase and ethanol inhibition analyses of protein processing in E. coli cells and minicells, respectively, indicated that processing of the FlaA precursor was incomplete. Native and recombinant FlaA were identical as assessed by antibody reactivity and sodium dodecyl sulfate- and two-dimensional polyacrylamide gel electrophoretic mobilities. Soluble FlaA was not detected in either the cytoplasmic or the periplasmic fractions of E. coli transformants. Fractionation of E. coli cell envelopes unexpectedly revealed that FlaA precursor and FlaA were associated with both the cytoplasmic and outer membranes. This is the first report of expression in E. coli of a T. pallidum protein which could not be cloned or expressed with its native promoter. Our data also indicate that information obtained in E. coli regarding the subcellular location of cloned treponemal proteins must be cautiously extrapolated to T. pallidum.

Structural studies of the contractile tail sheath protein of bacteriophage T4. 2. Structural analyses of the tail sheath protein, gp18, by limited proteolysis, immunoblotting and immunoelectron microscopy

The molecular structure of the T4 phage tail sheath protein, gp18, was studied by limited proteolysis, immunoblotting, and immunoelectron microscopy. Gp18 is extremely resistant to proteolysis in the assembled form of either extended or contracted sheaths, but it is readily cleaved by proteases in the monomeric form, giving rise to stable protease-resistant fragments. Limited proteolysis with trypsin gave rise to a trypsin-resistant fragment, Ala82-Lys316, with a molecular weight of 27K. Chymotrypsin- and thermolysin-resistant fragments were also mapped close to the trypsin-resistant region. The time course of trypsin digestion of the monomeric gp18 as monitored by SDS-polyacrylamide gel electrophoresis and immunoblotting of the gel revealed that the polypeptide chain consisting of 658 amino acid residues is sequentially cleaved at several positions from the C terminus. The N-terminal portion, Thr1-Arg81, was then removed to form the trypsin-resistant fragment. Immunoelectron microscopy revealed that the polyclonal antibodies against the trypsin-resistant fragment bound to the tail sheath. This supported the idea that at least part of the protease-resistant region of gp18 constitutes the protruding part of the sheath protein as previously revealed with three-dimensional image reconstruction from electron micrographs by Amos and Klug [Amos, L. A., & Klug, A. (1975) J. Mol. Biol. 99, 51-73].

Structural studies of the contractile tail sheath protein of bacteriophage T4. 1. Conformational change of the tail sheath upon contraction as probed by differential chemical modification

Differential chemical modifications of tyrosine residues of the tail sheath protein, gp18, were performed to elucidate the structural change of the tail sheath upon contraction. Tyrosine residues of monomeric gp18, extended tail sheath, and contracted tail sheath were nitrated by tetranitromethane, and the modified tyrosine residues in each state of the sheath protein were identified by peptide mapping and amino acid sequence analyses of the isolated peptides. Of 31 tyrosine residues in gp18 monomer or in the extended sheath, 12 or 13 residues (Tyr63 and/or -73, -225, -254, -270, -304, -455, -460, -493, -532, -535, -569, and -590) were modified. When photo-CIDNP difference spectra were measured with monomeric gp18, two peaks, which are due to highly exposed tyrosine residues on the molecular surface of gp18, were observed. These two peaks disappeared when the monomeric gp18 was nitrated. With contracted sheath, however, only eight tyrosine residues (Tyr225, -254, -270, -455, -460, -493, -532, and -535) were nitrated on the contracted sheath. Chemical modification of cysteine residues by sulfhydryl group specific reagent ABD-F [(4-aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole] revealed that, among five cysteine residues, Cys377, Cys477, and Cys607 have a sulfhydryl group. Cys402 and Cys406 were modified only under reducing conditions, which strongly suggested the presence of a disulfide bond between these two residues.

Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum.

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a lambda gt11 clone (designated lambda A34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of the entire protein was selected from a T. pallidum(pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pR14 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

Genetic comparison of bacteriophage PS17 and Pseudomonas aeruginosa R-type pyocin

PS17 is a bacteriophage of Pseudomonas aeruginosa that is serologically cross-reactive with phage tail-like bacteriocins called R-type pyocins. In addition to having immunological cross-reactivity, certain genes are functionally complementable between PS17 and R-type pyocins. To compare the genetic structures of PS17 and R-type pyocins, a physical map of PS17 genes was constructed by cloning phage DNA fragments on RSF1010-derived vector plasmids. The head and tail gene clusters were tandemly arrayed and together occupied about half of the 41-kilobase-pair PS17 chromosome. With use of these phage clones, the following results were obtained with respect to the genetic relationship between PS17 and R-type pyocins: (i) serological cross-reaction between PS17 and pyocin occurred for the major sheath protein and two components of the fiber, (ii) a certain pyocin mutation was complemented by cloned phage fragments, and (iii) the phage DNA fragment carrying sheath and core tube genes was shown to hybridize to the DNA fragment carrying the pyocin R2 genes.

Nucleotide sequence of the tail sheath gene of bacteriophage T4 and amino acid sequence of its product.

The nucleotide sequence of gene 18 of bacteriophage T4 was determined by the Maxam-Gilbert method, partially aided by the dideoxy method. To confirm the deduced amino acid sequence of the tail sheath protein (gp18) that is encoded by gene 18, gp18 was extensively digested by trypsin or lysyl endopeptidase and subjected to reverse-phase high-performance liquid chromatography. Approximately 40 peptides, which cover 88% of the primary structure, were fractionated, the amino acid compositions were determined, and the corresponding sequences in DNA were identified. Furthermore, the amino acid sequences of 10 of the 40 peptides were determined by a gas phase protein sequencer, including N- and C-terminal sequences. Thus, the complete amino acid sequence of gp18, which consists of 658 amino acids with a molecular weight of 71,160, was determined.

Affinity chromatography on immobilized anhydrotrypsin: general utility for selective isolation of C-terminal peptides from protease digests of proteins.

Recently we have succeeded in the efficient isolation of the C-terminal peptides from tryptic digests of the tail sheath protein (with C-terminal Gly) and the tube protein (with C-terminal Glu) of bacteriophage T4, by taking advantage of a unique property of immobilized anhydrotrypsin, that is, a strong specific affinity for peptides containing Arg or Lys residues at their C-termini. In this study, the utility of affinity chromatography on immobilized anhydrotrypsin was further demonstrated in the cases of Streptomyces subtilisin inhibitor (as a reduced and S-carboxymethylated form, with C-terminal Phe) and alpha 1-antitrypsin (with C-terminal Lys). By subjecting a tryptic digest of the former protein and a chymotryptic digest of the latter protein to the affinity chromatography, the C-terminal peptides were specifically recovered in the breakthrough fraction and in the adsorbed fraction, respectively. It was further shown that immobilized anhydrotrypsin can also adsorb peptides with C-terminal S-aminoethyl-Cys residues and exerts adsorptive ability even toward the peptides in solution containing urea at a high concentration if appropriate precautions are taken. These findings suggest the general utility of this simple method for C-terminal peptide isolation, which is extremely helpful for studies to confirm amino acid sequences deduced from nucleotide sequences of the cDNA (or genomic DNA) of proteins.

Primary structure of the tail sheath protein of bacteriophage T4 and its gene

Complete sequence determination of gene 18 encoding the tail sheath protein was carried out mainly by the Maxam-Gilbert method. Approximately 40 peptides contained in a tryptic digest and a lysyl endopeptidase digest of gp 18 were isolated by reversed-phase high-performance liquid chromatography. All the peptides were identified along the nucleotide sequence of gene 18 based on the amino acid compositions. These peptides cover 88% of the total primary structure. Furthermore, the amino acid sequences of 9 of the 40 peptides were determined by a gas-phase protein sequencer; one of them turned to be the N-terminal one. The C-terminal peptide in the tryptic digest was isolated from the unadsorbed fraction of affinity chromatography on immobilized anhydrotrypsin and the amino acid sequence was also determined. Thus, the complete primary structure of gp 18 was determined; it has 658 amino acid residues and a molecular weight of 71,160.

Contractile tail sheath protein of bacterio-phage T4.

Tail sheath protein of phage T4 constitutes the outer cylinder of the co-cylindrical structure of the tail, which appears to have evolved for efficient infection of the phage.It consists of 144 protomer molecules arranged in 24 annuli. Contraction of the sheath is exothermic and irreversible. Recently, primary structure of the protomer tail sheath protein (gp18) was determined from nucleotide sequence of the gene, which was subsequently confirmed by peptide analysis of the gene product. Use of the primary structure and available gene 18 mutants for further studying structure-function relation of the gene product and the mechanism of sheath contraction are discussed.

Sheath disassembly in Methanospirillum hungatei strain GP1

Sheaths of Methanospirillum hungatei are very resilient structures and consist of circumferential rings, which have been likened to the hoops of a barrel. The isolated sheaths are dramatically disassembled to the individual hoops upon treatment with β-mercaptoethanol at 90 °C. Sheath disassembly resulted in a large decrease in suspension turbidity and a release of approximately 10% of sheath protein in the form of 4.6 to 7.0 kiloDalton (kDa) peptides. These are referred to as "glue peptides" to suggest a function in linking the hoops together to form the intact sheath. The liberated hoops consisted of a protein surface array crystallized on a matrix material. Intact sheaths, or hoops largely freed of the glue peptides, could be solubilized at 90 °C by (i) a combination of β-mercaptoethanol and sodium dodecyl sulfate, (ii) by alkaline conditions of pH 12 or more, or partially (iii) by carbonate anion at pH 11. Fractionation by liquid chromatography of hoops solubilized by alkaline conditions of pH 12.6 revealed major polypeptides of about 24 and 45 kDa; a smaller peak occurred at 12 kDa. Based on the dimensions of the unit cell of the crystalline array, we suggest that two copies of the 24-kDa polypeptide form the 5.6 × 2.8 nm unit cell as a dimer, which then tends to form larger aggregates. The 2.8-nm subunit may be a dimer of the 12-kDa species. Sheaths of M. hungatei strain JF1 exhibited a similar polypeptide profile, but in this case, the 45-kDa species predominated.