Three truncated forms of serum albumin associated with pancreatic pseudocyst

Plasma from a patient with chronic pancreatic pseudocyst showed an additional more negative albumin band (18%) on agarose gel electrophoresis. Both components bound (63)Ni(2+), indicating intact N-terminals; however, electrospray ionisation analysis of the intact proteins showed the mass of more negative albumin was 1254 Da less than the control and that the apparently normal band was 112 Da less. Reverse phase mapping and mass analysis of CNBr peptides showed three proteolytically modified forms of the C-terminal peptide indicating that some 81% of the albumin molecules lacked the C-terminal Leu residue, that 18% lacked the C-terminal KKLVAASQAALGL and that approximately 1% lacked the QAALGL sequence. These findings were further verified by tryptic mapping of the aberrant CNBr peptides. The truncations probably result from exposure of the albumin to 'leaking' pancreatic endo and exoproteases. During less acute phases of the disease, the 13 and 6 residue truncated forms together decreased to less than 1%, while the des-Leu(585) form made up the balance; no normal albumin was detected. This suggested that the des-Leu(585) form might be present at low levels in the plasma of normal individuals and CNBr mapping confirmed that it constituted 4-15% of the albumin from normal plasma.     

Albumin Rugby Park: a truncated albumin variant caused by a G → C splice-site mutation in nitro 13

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G → C mutation at oposition 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.     

Albumin Rugby Park: a truncated albumin variant caused by a G-->C splice-site mutation in intron 13.

Three members of a family were found to be heterozygous for a fast albumin variant (Albumin Rugby Park) that made up only 8% of total serum albumin. Isoelectric focussing indicated an increased negative charge on the C-terminal CNBr peptide and C-terminal sequence analysis of the native protein showed an aberrant sequence of -Ser-Phe. Sequence analysis of PCR-amplified DNA indicated a G-->C mutation at position 1 of the 13th intron and this was confirmed by restriction digestion. The replacement of the obligate GT sequence by CT at the exon/intron boundary prevents splicing of the 13th intron and translation continues for 21 nucleotides until a stop codon is reached. The new protein lacks the 14 amino acids coded for in the 14th exon (GKKLVAASQAALGH), but these are replaced by 7 new residues (LLQFSSF), giving a truncated albumin of 578 residues.     

Structural characterization of a glycoprotein variant of human serum albumin: albumin Casebrook (494Asp → Asn)

Albumin Casebrook is an electrophoretically slow genetic variant of human albumin with a relative molecular mass 2.5 kDa higher than normal albumin. It constitutes about 35% of total serum albumin in heterozygous carriers. The decrease in negative charge observed on incubation with sialidase suggested the presence of a carbohydrate moiety and the normalization of molecular weight following treatment with Endo-F indicated that this was an N-linked oligosaccharide. Partial acid hydrolysis and limited tryptic digestion established that the oligosaccharide was located in the C-terminal domaine, between residues 367 and 585. Tryptic, chymotryptic and S. aureus V8 proteinase digestions were carried out and the resulting glycopeptides were purified on concanavalin A-Sepharose. Peptide mapping of bound and unbound fractions followed by amino acid composition and sequence analysis, established a point mutation of 494 Asp → Asn. This introduces an Asn-Glu-Thr N-linkrf oligosaccharide attachment sequence centered on Asn-494 and explains the increase in molecular mass. There was no apparent pathology associated with the presence of this new glycosylated albumin, which was detected in two unrelated individuals of Anglo-Saxon descent.     

Butyrivibriocin AR10, a new cyclic bacteriocin produced by the ruminal anaerobe Butyrivibrio fibrisolvens AR10: characterization of the gene and peptide

The gene (bviA) encoding the ruminal bacteriocin butyrivibriocin AR10 was cloned from an EcoRI library by using an oligonucleotide probe based on a partial peptide sequence of the previously isolated peptide. The gene encoded an 80 amino acid prebacteriocin that demonstrated significant identity with the cyclic bacteriocin gassericin A. Negative ion time of flight mass spectroscopic analysis (ESI/MS) indicated a mass of 5981.5 Da for the isolated bacteriocin, a molecular mass that could not be generated by removal of a leader peptide alone. However, an N- to C-terminal cyclization of the predicted mature bacteriocin resulted in a peptide that conformed to the determined mass and charge characteristics. Northern blotting confirmed that expression of bviA mirrored the production of the bacteriocin in both liquid and solid media.     

Amino-acid sequence of the smallest protein of the cytochrome c1 subcomplex from beef heart mitochondria

A cytochrome c1 subcomplex was obtained from purified complex III. The subcomplex consists of three protein subunits, present at an equimolar stoichiometry. The primary structures of two of these proteins, the heme-carrying cytochrome c1 and a protein with a molecular mass of 9175 Da, have been published by Wakabayashi et al. The covalent structure of the smallest subunit is presented in this paper. This protein consists of 62 amino acid residues; its molecular mass was calculated to be 7189 Da. The sequence was determined by complete solid-phase Edman degradation of the uncleaved polypeptide and was confirmed by sequencing the C-terminal fragment resulting from cleavage of a single tryptophyl bond.     

Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24–70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250–430) likely forms a 16-strand β-barrel and appears to be related to the unusual N-terminal SLH-domain-containing β-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.     

Identification of an insecticidal crystal protein from Bacillus thuringiensis DSIR517 with significant sequence differences from previously described toxins.

The nucleotide (nt) sequence of a DNA segment containing the majority of a gene cloned from Bacillus thuringiensis DSIR517 encoding a 130 kDa insecticidal crystal protein has been determined. Sequence analysis reveals an open reading frame (ORF) of 3453 nt. The ATG initiation codon, which is preceded by a potential ribosome-binding site sequence, was confirmed by N-terminal amino acid sequencing. The ORF extends beyond the 3' terminus of the cloned fragment; however, the high degree of homology between the deduced amino acid sequence of this ORF and other Cry proteins suggests the clone lacks only five C-terminal amino acids. Making this assumption, the ORF of 3468 nt encodes a protein of 1156 amino acids with an estimated molecular mass of 129700 Da. Analysis of the deduced amino acid sequence reveals a number of features characteristic of Cry proteins. Alignment of the Cry 517 protein sequence with other Cry proteins suggests it is most closely related to the cryIA-E genes but sufficiently different to form a new cryI gene subclass.     

A new chemical approach to differentiate carboxy terminal peptide fragments in cyanogen bromide digests of proteins

We present a novel approach to perform C-terminal sequence analysis by discriminating the C-terminal peptide in a mass spectral analysis of a CNBr digest. During CNBr cleavage, all Met-Xxx peptide bonds are cleaved and the generated internal peptides all end with a homoserine lactone (hsl)-derivative. The partial opening of the hsl-derivatives, by using a slightly basic buffer solution, results in the formation of m/z doublets (Δm = 18 Da) for all internal peptides and allows to identify the C-terminal peptide which appears as a singlet in the mass spectra. Using two model proteins we demonstrate that this approach can be applied to study proteins purified in gel or in solution. The chemical opening of the hsl-derivative does not require any sample clean-up and therefore, the sensitivity of the C-terminal sequencing approach is increased significantly. Finally, the new protocol was applied to characterize the C-terminal sequence of two recombinant proteins. Tandem mass spectrometry by MALDI-TOF/TOF allowed to identify the sequence of the C-terminal peptides. This novel approach will allow to perform a proteome-wide study of C-terminal proteolytic processing events in a high-throughput fashion.     

Kunitz-type proteinase inhibitors derived by limited proteolysis of the inter-alpha-trypsin inhibitor, IV. The amino acid sequence of the human urinary trypsin inhibitor isolated by affinity chromatography

An acid-resistant trypsin inhibitor from human urine and serum is released in vivo by limited proteolysis from the high molecular acid-labile inter-alpha-trypsin inhibitor. The inhibitor shows an apparent molecular mass of 30 000 Da and is composed of two Kunitz-type domains. The domains are released in vitro by prolonged tryptic hydrolysis. The C-terminal domain is responsible for antitryptic activity. For the other domain no inhibitory activity towards proteinases, i.e. chymotrypsin, trypsin, pancreatic and leucocytic elastase has been demonstrated so far. The polypeptide chain comprising both domains consists of 122 residues and has a molecular mass of only 13 400 Da. In this work we have found that both, the N-terminal extension peptide with 21 residues and the "inactive" domain are linked O-glycosidically and N-glycosidically, respectively, with large carbohydrate moieties. The N-terminal amino acid sequence of the human urinary trypsin inhibitor was determined by solid-phase Edman degradation of a single peptide. The molecular mass calculated for the total polypeptide chain of 143 residues should be 15 340 Da; from the difference to the measured value (30 000 Da) it is concluded that the glycopeptide contains a considerable carbohydrate moiety.     

Full activation of Enterococcus faecalis gelatinase by a C-terminal proteolytic cleavage

Enterococci account for nearly 10% of all nosocomial infections and constitute a significant treatment challenge due to their multidrug resistance properties. One of the well-studied virulence factors of Enterococcus faecalis is a secreted bacterial protease, termed gelatinase, which has been shown to contribute to the process of biofilm formation. Gelatinase belongs to the M4 family of bacterial zinc metalloendopeptidases, typified by thermolysin. Gelatinase is synthesized as a preproenzyme consisting of a signal sequence, a putative propeptide, and then the mature enzyme. We determined that the molecular mass of the mature protein isolated from culture supernatant was 33,030 Da, which differed from the predicted molecular mass, 34,570 Da, by over 1,500 Da. Using N-terminal sequencing, we confirmed that the mature protein begins at the previously identified sequence VGSEV, thus suggesting that the 1,500-Da molecular mass difference resulted from a C-terminal processing event. By using mutants with site-directed mutations within a predicted C-terminal processing site and mutants with C-terminal deletions fused to a hexahistidine tag, we determined that the processing site is likely to be between residues D304 and I305 and that it requires the Q306 residue. The results suggest that the E. faecalis gelatinase requires C-terminal processing for full activation of protease activity, making it a unique enzyme among the members of the M4 family of proteases of gram-positive bacteria.     

Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

A cecropin-like antimicrobial peptide, Gm cecropin, was purified from hemolymph of larvae of the wax moth, Galleria mellonella, immunized against E. coli, and its antibacterial activity was examined in a radial diffusion assay. The molecular mass of Gm cecropin was 4,160.69 Da by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis. The full-length cDNA of the Gm cecropin precursor was cloned by a combination of RT-PCR, based on the N-terminal sequence obtained by Edman degradation, and 5'-RACE-PCR. Analysis of the cDNA showed that cecropin is synthesized as a prepropeptide, with a putative 22-residue signal peptide, a 4-residue propeptide and a 39-residue mature peptide with a calculated mass of 4,344.18 Da the difference between the calculated and measured masses suggests that Gm cecropin is a 37-residue peptide generated by removal of the C-terminal residue and amidation.     

A Kazal-type trypsin inhibitor from the protochordate Ciona intestinalis.

A trypsin inhibitor from Ciona intestinalis, present throughout the animal, was purified by ion-exchange chromatography followed by four HPLC steps. By MS the molecular mass of the native form was determined to be 6675 Da. The N-terminal amino acid sequence was determined by protein sequencing, but appeared to be partial because the theoretical molecular mass of the protein was 1101 Da too low. Thermolysin treatment gave rise to several fragments each containing a single disulphide bridge. By sequence analysis and MS intramolecular disulphide bridges could unequivocally be assigned to connect the pairs Cys4-Cys37, Cys8-Cys30 and Cys16-Cys51. The structure of the inhibitor is homologous to Kazal-type trypsin inhibitors. The inhibitor constant, KI, for trypsin inhibition was 0.05 nM whereas chymotrypsin and elastase were not inhibited. To reveal the complete sequence the cDNA encoding the trypsin inhibitor was isolated. This cDNA of 454 bp predicts a protein of 82 amino acid residues including a 20 amino acid signal peptide. Moreover, the cDNA predicts a C-terminal extension of 11 amino acids compared to the part identified by protein sequencing. The molecular mass calculated for this predicted protein is in accordance with the measured value. This C-terminal sequence is unusual for Kazal-type trypsin inhibitors and has apparently been lost early in evolution. The high degree of conservation around the active site strongly supports the importance of the Kazal-type inhibitors.     

Cloning and sequencing of the central region of the flagellin gene from the Gram-positive bacterium Clostridium tyrobutyricum ATCC 25755

The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum fiagellin gene to improve the immunoenzymatic counting of cells after milk filtration. The coding region was amplified by PCR, and the amplified products were cloned. A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da. The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain. A PCR-restriction fragment length polymorphism analysis of amplified C. tyrobutyricum flagellin gene products confirmed the variability of the central domain. The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA. Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation.     

Heterogeneous post-translational modification of Actinobacillus actinomycetemcomitans fimbrillin

Fresh isolates of Actinobacillus actinomycetemcomitans produce bundle-forming fimbriae. The exact molecular mass of A. actinomycetemcomitans fimbrillin, a structural subunit of fimbriae, was determined by liquid chromatography-electrospray ionization mass spectrometry. Three major molecular species with 6,226.0, 6,366.0, and 6,513.0 Da were detected in a purified fimbrial fraction from the strain 310-a. These molecular masses were significantly higher than the molecular weight (5,118 Da) calculated from nucleotide sequence data of the fimbrillin gene, flp, suggesting that the fimbrial peptides were post-translationally modified. Modification of the fimbrial peptides was also suggested by an N-terminal amino acid sequence analysis of fimbrillin peptic fragments, with the modified amino acids being due to seven serine or asparagine residues located in the C-terminal region. A periodate oxidation/biotin-hydrazide labeling assay of fimbrillin suggested that it might be glycosylated.     

Demonstration by Mass Spectrometry that Purified Native Treponema pallidum Rare Outer Membrane Protein 1 (Tromp1) Has a Cleaved Signal Peptide

Purified native Tromp1 was subjected to mass spectrometric analysis in order to determine conclusively whether this protein possesses a cleaved or uncleaved signal peptide. The molecular masses of Tromp1, three Treponema pallidum lipoproteins, and a bovine serum albumin (BSA) control were determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. The molecular masses of all of the T. pallidum lipoproteins and BSA were within 0.7% of their respective calculated masses. The molecular mass of Tromp1 was 31,510 Da, which is consistent with a signal-less form of Tromp1, given a calculated mass of unprocessed Tromp1 of 33, 571 Da, a difference of 2,061 Da (a 6.5% difference). Purified native Tromp1 was also subjected to MALDI-TOF analysis in comparison to recombinant Tromp1 following cyanogen bromide cleavage, which further confirmed the identity of Tromp1 and showed that native Tromp1 was not degraded at the carboxy terminus. These studies confirm that Tromp1 is processed and does not contain an uncleaved signal peptide as previously reported.     

Cloning and sequencing of the gene encoding X-prolyl-dipeptidyl aminopeptidase (PepX) from Streptococcus thermophilus strain ACA-DC 4

AIMS: To clone and sequence the pepX gene from Streptococcus thermophilus. METHODS AND RESULTS: Three pairs of primers were used in polymerase chain reactions using as template the total DNA from Strep. thermophilus ACA-DC 4 in order to amplify, clone and sequence the pepX gene. Sequence analysis revealed an open reading frame of 2268 nucleotides encoding a protein of 755 amino acids. The calculated molecular mass of 85 632 Da agreed well with the apparent molecular mass of 80 000 Da previously determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration for the monomeric form of the purified enzyme. CONCLUSIONS: The pepX gene from Strep. thermophilus ACA-DC 4 was cloned and sequenced. The PepX protein showed significant sequence similarity with PepX enzymes from other lactic acid bacteria and contained a motif which was almost identical with the active site motif of the serine-dependent PepX family. SIGNIFICANCE AND IMPACT OF THE STUDY: There are economic and technological incentives for accelerating and controlling the process of cheese ripening. To achieve this, starters may be modified by introducing appropriate genes from other food-grade bacteria. New or additional peptidase activities may alter or improve the proteolytic properties of lactic acid bacteria.