Identification of a novel heterodimeric outer membrane protein of Porphyromonas gingivalis by two-dimensional gel electrophoresis and peptide mass fingerprinting.

Porphyromonas gingivalis is a Gram-negative, anaerobic bacterium associated with chronic periodontitis. A 2D electrophoretic analysis of the outer membrane of P. gingivalis W50 revealed a dominant train of spots at 40-41 kDa. The proteins in the train of spots were digested in-gel with trypsin and identified by MS. The train of spots represented two proteins, designated Omp40 and Omp41 that share 47% sequence identity. Preparation of outer membranes in the absence of protease inhibitors resulted in partial cleavage of Omp40 and Omp41 to produce an N-terminal and C-terminal fragment of both proteins. The N-terminal fragments displayed the same isoelectric heterogeneity as the intact proteins. Almost 100% of the amino-acid sequence of these N-terminal fragments in each 2D gel spot was verified suggesting lack of post-translational modification. Re-subjecting a single N-terminal domain spot to 2D electrophoresis resulted in the complete series of spots being reproduced, suggesting that the heterogeneity was related to conformational equilibria. Under reduced conditions and without heating, Omp40 and Omp41 migrated as 34- to 35-kDa proteins in SDS/PAGE whereas under nonreduced conditions the proteins migrated as 70-kDa proteins, suggesting the formation of dimers through intersubunit disulfide bonds. The proteins each contain two cysteine residues in the conserved sequence RPVSCPECPE. Tryptic peptides generated from the nonreduced forms of the proteins confirmed the presence of heterodimers stabilized through intersubunit disulfide bond formation. With the exception of heterodimer formation, the two proteins share several similarities with OmpA-like porins of other Gram-negative bacteria including consensus sequence, abundance, modification by heat, overall length and positioning of domains.

Kappacin, a novel antibacterial peptide from bovine milk.

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk kappa-casein composed of glycosylated and phosphorylated forms of different genetic variants. We have demonstrated that CMP has growth-inhibitory activity against the oral opportunistic pathogens Streptococcus mutans and Porphyromonas gingivalis and against Escherichia coli. CMP was fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC), and each fraction was tested for activity against S. mutans in a 96-well-plate broth assay. Fractions were characterized by N-terminal sequence analysis and mass spectrometry. The active form of CMP was shown to be the nonglycosylated, phosphorylated kappa-casein (residues 106 to 169) [kappa-casein(106--169)], which we have designated kappacin. Endoproteinase Glu-C was used to hydrolyze CMP, and the generated peptides were separated using RP-HPLC and gel filtration-HPLC and then tested for activity against S. mutans. The peptide Ser(P)(149)kappa-casein-A(138--158) was the only peptide generated by endoproteinase Glu-C digestion that exhibited growth-inhibitory activity. Peptides corresponding to the sequences of the inhibitory peptide Ser(P)(149)kappa-casein-A(138--158) and its nonphosphorylated counterpart kappa-casein-A(138--158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P)(149) kappa-casein-A(138--158) displayed growth-inhibitory activity against S. mutans (MIC, 59 microg/ml [26 microM]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.

MALDI-PSD-MS analysis of the phosphorylation sites of caseinomacropeptide.

Caseinomacropeptide (CMP) is a 64 amino acid polypeptide corresponding to kappa-casein 106-169. CMP naturally exists in several forms due to extensive posttranslational modifications including glycosylation and phosphorylation. The aglycosylated, phosphorylated form of CMP has been shown to exhibit antibacterial activity. The aim of this study was to use matrix assisted laser desorption/ionization post source decay mass spectrometry (MALDI-PSD-MS) to identify the phosphorylation sites in the CMP sequence. CMP was isolated from a chymosin digest of casein by HPLC and then digested with endoproteinase Glu-C to generate peptides suitable for MALDI-PSD-MS analysis. This analysis showed that CMP is fully phosphorylated at Ser(149) and only partially phosphorylated at Ser(127.) Dehydroalanyl residues corresponding to the phosphoserines of CMP were detected upon MALDI-PSD-MS analysis suggesting that the phosphoryl bond in phosphoserine is very labile during PSD analysis such that the phosphoryl group may be lost before backbone fragmentation.

Quantitative and qualitative influences of tapasin on the class I peptide repertoire.

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta(2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

N-terminal sequence analysis of bovine dentin phosphophoryn after conversion of phosphoseryl to S-propylcysteinyl residues.

Bovine dentin phosphophoryn (BDP), a protein rich in aspartyl (Asp) and O-phosphoseryl (Ser[P]) residues, is synthesized by odontoblasts and is believed to be involved in matrix-mediated biomineralization of dentin. We have purified BDP, using selective precipitation and ion exchange chromatography, from an EDTA soluble dentin extract and converted the Ser(P) residues to S-propylcysteinyl residues that are stable to Edman degradation, facilitating the determination of the amino acid sequence of the N-terminal 38 residues. After the initial Asp-Ser(P)-Pro-Asn-Ser(P)-Ser(P)-Asp-Glu-Ser(P)-Asn-Gly-, the sequence contained the repeated motifs Asp-Ser(P) and Asp-Ser(P)-Ser(P). Purified BDP migrated as a single band on gradient SDS-PAGE with an apparent molecular weight of 156 kDa. This value was consistent with the molecular weight of the dephosphorylated protein of 105 kDa determined by means of MALDI mass spectrometry.

Foetal fluid balance and hormone status following nephrectomy in the foetal sheep.

1. The role of the kidneys in the maintenance of normal foetal plasma (FP) composition and hormone concentrations was examined in the present study. Five ovine foetuses were chronically cannulated and nephrectomized (nephx) at 100 +/- 1 days of gestation and maintained for 14 days. These were compared to five intact control foetuses. 2. Four hours after nephx, FP renin concentrations were significantly lower than in control foetuses. By 48 h, renin concentrations in nephx foetuses were below the level of detectability of the assay. Foetal plasma aldosterone concentrations declined in nephx foetuses, but were not significantly different to those in control foetuses (P = 0.08). 3. During the second week, the nephx foetuses were significantly hypoxic, but FP erythropoietin concentrations were not increased. Adrenocorticotropic hormone (ACTH) and cortisol concentrations, when measured on day 14, were not different between the two groups. Adrenocorticotropic hormone levels were correlated with adrenal weight at post-mortem. 4. Foetal plasma creatinine, magnesium and phosphate concentrations in nephx foetuses increased, eventually reaching values approximately twice that in controls. Foetal plasma chloride levels decreased continuously in nephx foetuses, eventually being 23 mmol/L lower than controls. Maternal plasma composition was unchanged. 5. Total foetal fluid (amniotic + allantoic) volumes were reduced when measured at post-mortem on day 14 after nephx. The composition of both fluids was significantly altered in the nephx foetuses compared with controls. 6. Fetuses can survive in utero for 2 weeks after bilateral nephrectomy. However, there are multiple changes in plasma composition that may compromise foetal survival in the long term.

Developments in matrix-assisted laser desorption/ionization peptide mass spectrometry.

Most characteristics of matrix-assisted laser desorption/ionization (MALDI) are ideal for the analysis of biomolecules. New preparation techniques have dramatically increased mass accuracy and resolution, making MALDI a high-performance mass spectrometric technique for peptide mass analysis. Attempts to obtain amino acid sequence information by MALDI have been partially successful. The technique has been put to novel uses in protein primary structure characterization.

Solid phase synthesis and biological activity of rat relaxin.

The peptide hormone relaxin was isolated in good yield from the ovaries of the pregnant rodent Rattus rattus using a simplified purification schedule. It was subjected to comprehensive chemical characterization to confirm both its purity and predicted composition. The peptide was also chemically synthesized by the solid phase procedure. The two chains comprising the hormone were each assembled by the Boc-polystyrene method and, following conventional purification, combined in solution to form the single intramolecular and two intermolecular disulfide bonds. Following purification, the synthetic rat relaxin was fully chemically characterized and shown to be indistinguishable from the native peptide including by secondary structure analysis using circular dichroism spectroscopy. The native and synthetic rat relaxins were shown to be equally biologically active in the in vitro rat uterine relaxation assay and had pEC50 values that were comparable to synthetic human H2 relaxin.

Aspects of the sequencing of carbohydrates and oligonucleotides by matrix-assisted laser desorption/ionization post-source decay.

The possibility of structural elucidation of carbohydrates and oligonucleotides by matrix-assisted laser desorption/ionization followed by post-source decay (MALDI-PSD) is investigated. Spectra of both classes of compounds exhibit the better signal-to-noise ratios for the intact species in the negative-ion mode, but the most informative spectra for structural elucidation by PSD are obtained in the positive-ion mode. A novel fragmentation mode is demonstrated on a model sialylated complex type N-linked oligosaccharide. Prompt fragmentation in the positive-ion mode can remove the sialic acids at high laser irradiance if a suitable matrix is used. The remaining sugar backbone is then characterized by post-source decay. A modified oligonucleotide is analyzed with 3-hydroxypicolinic acid as the matrix and ammonium tartrate to displace sodium. Enough structural information is obtained to verify the modification.

Amino acid residues 721-729 are required for full factor VIII activity.

Recombinant two-chain factor VIII, from which the B domain had been deleted, was expressed in Chinese hamster ovary cells. In addition to the major product, three minor factor VIII forms were isolated. The A2 domains generated by thrombin cleavage showed different electrophoretic mobilities. Peptide mapping of the A2 domains showed that two of the factor VIII forms had the expected C-terminus of the heavy chain at Arg740 [FVIII-(1-740)] and that the other factor VIII forms had C-termini at Tyr729 [FVIII-(1-729)] or Glu720 [FVIII-(1-720)]. The major FVIII-(1-740) form, FVIII-(1-729), and FVIII-(1-720) contained sulfated tyrosine residues at Tyr718, Tyr719 and Tyr723. The minor FVIII-(1-740) form was shown to lack sulfation at these positions. The specific clotting activity was approximately 1 x 10(4) U/mg for FVIII-(1-740) (both forms) and FVIII-(1-729), but twofold lower for FVIII-(1-720). A time study of thrombin activation showed that FVIII-(1-720) was activated slower than FVIII-(1-740), FVIII-(1-729) and plasma-derived factor VIII. Partially sulfated FVIII-(1-740) was activated at the same rate as the fully sulfated FVIII-(1-740). The equilibrium dissociation constant for binding of factor VIII to inactivated immobilized thrombin was the same for all factor VIII forms, showing that the slower activation of FVIII-(1-720) was not due to a lower affinity for the anion-binding exosite in thrombin.

Refolding of bovine pancreatic trypsin inhibitor via non-native disulphide intermediates.

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species. The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30-51, 5-14) and (30-51, 5-38). Moreover, the rates of folding through each of these intermediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathway, and their roles are readily explicable on the basis of their conformations.

Rabbit skeletal muscle alpha alpha-tropomyosin expressed in baculovirus-infected insect cells possesses the authentic N-terminus structure and functions.

When expressed in E. coli, skeletal muscle alpha-tropomyosin has an unacetylated N-terminus. Unacetylated alpha-tropomyosin lacks important functions; this is non-polymerizable and has a low affinity to actin. In the present work, in order to obtain fully functional recombinant alpha-tropomyosin, rabbit skeletal muscle alpha-tropomyosin (alpha-tropomyosin BV) has been expressed in baculovirus-infected insect cells. alpha-TropomyosinBV was not distinguishable from the authentic tropomyosin, not only in functional properties but also in blocked N-terminus. To know the N-terminus structure of alpha-tropomyosinBV, the N-terminal segment six amino acids long, MDAIKK, has been specifically and efficiently removed from alpha-tropomyosinBV by use of an immobilized proteolytic enzyme system based on E. coli cell bodies which carry the ompT gene product, a proteolytic enzyme localized on the outer cell wall of E. coli. The structure of recombinant alpha-tropomyosinBV was shown to be identical to the authentic protein by electrospray mass spectrometry and protein sequencing analysis. Additionally, electrospray mass spectrometry indicated a single phosphorylation not only in alpha- but also beta-tropomyosin chains in the rabbit skeletal muscle. The differentiated susceptibilities of potential ompT cleavage sites are indicative of a non-coiled-coil conformation of the N-terminus of alpha-tropomyosin.

Oxidation of peptides during electrospray ionization.

[M + H + 16]+ ions were observed in the electrospray ionization mass spectra of several synthetic and naturally occurring peptides. Initial results have shown that the appearance of the modification is dependent on the field strength at the electrospray needle and the flow rate of the solution passing through the capillary. Mass spectrometric experiments on peptides showing the + 16 Da modification attributed the change to the selective oxidation of either a methionyl, tryptophanyl, or tyrosyl residue present in the peptide. These results were further confirmed by tandem mass spectrometry experiments on peptides with a methionyl residue at either the N- or C-terminus, or within the peptide chain. This effect can occur under normal operating conditions and therefore care must be taken in the analysis of samples containing these oxidizable residues. Conversely, the selectivity of the oxidative process may be used to enhance the information obtained from the mass spectrometric analysis. For example, we show results for the analysis of a tryptic digest of the protein myoglobin where the occurrence of the [M + H + 16]+ ion is used, along with the molecular weight data, to correctly identify the trypic fragment.

Two cysteines, two histidines, and one methionine are ligands of a binuclear purple copper center.

Cytochrome c oxidase contains a copper center, CuA, which is involved in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is comprised of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuration. Here we use a model of CuA, engineered into a structurally related but initially copperless protein, to study the structure of this copper center. The results from biochemical analysis, site-directed mutagenesis, and electrospray mass spectrometry support the binuclear model. Two cysteines, two histidines, and one methionine are the major ligands of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum. In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor perturbation of the optical spectrum.

Optimization of sample recovery from the nitrocellulose support used in plasma desorption mass spectrometry and its use for multiple analyses of insulin.

The parameters for recovery of sample from the nitrocellulose support used in plasma desorption are optimized. The losses in washing, in situ reactions, and extraction procedures are quantitatively evaluated. At least 80% of the sample can be effectively extracted or transferred to a polyvinyl difluoride membrane with 2-propanol-water mixture in the range 1:1-2:3 v/v. Quantitative losses of insulin during washing procedures vary from 0 to 50% depending on the washing procedure used. The losses in in situ reactions are negligible. Optimization of the procedures allows several successive procedures to be carried out after adsorption of 1 nmol of insulin on the nitrocellulose support. These include in situ reaction, extraction of A and B chains followed by S-alkylation, chain separation by high-performance liquid chromatography, mass spectrometric analysis of the separated chains, and finally automatic sequence after transfer to the sequenator.

Determination of the covalent structure of an N- and C-terminally blocked glycoprotein from endocuticle of Locusta migratoria. Combined use of plasma desorption mass spectrometry and Edman degradation to study post-translationally modified proteins.

The complete structure of protein isolated from endocuticle of sexually mature locusts, Locusta migratoria, has been determined by a combination of automatic Edman degradation and plasma desorption mass spectrometry. The protein is extensively post-translationally modified. The N-terminal is 5-oxoproline (pyroglutamic acid) and the C-terminal proline residue is amidated. Furthermore, the protein is glycosylated by a single N-acetyl-galactosamine residue at one, two or three threonines. The N-terminal sequence was obtained by analysing the N-acetylated N,O-permethylated derivative using plasma desorption mass spectrometry. The position and type of carbohydrate were determined by combining an HPLC-based carbohydrate analysis with the peak pattern of the phenylthiohydantoin derivative in automatic sequencing and with mass information on peptides. The protein has pronounced similarity to cuticular proteins from larvae of diptera and lepidoptera, but only slight resemblance to the previously sequenced locust exocuticular proteins. This indicates a similarity between soft larval cuticles and locust endocuticle, a similarity which may extend to their mechanical properties.

Sequence determination of N-acetylated-N,O-permethylated peptides by plasma desorption mass spectrometry.

The plasma desorption mass spectra of N-acetylated-N,O-permethylated peptides contain sufficient fragment ions to allow partial or complete sequence determination. By optimization of the derivatization procedure and an inclusion of a purification step by high-performance liquid chromatrography (HPLC) overall sensitivities on the high picomole level are obtained. By using limiting derivatization conditions the fully derivatized peptide is easily selected from the HPLC separation. Mainly N-terminal sequence ions are observed, facilitating sequence determination of naturally N-blocked peptides. Complete sequence determination of naturally formylated gramicidin A containing 15 amino acid residues and the naturally N-acetylated N-terminal heptapeptide from an acyl-CoA binding protein is demonstrated. The sequence of the first six N-terminal residues was obtained by derivatization of the A-chain of insulin.