The effect of deimination of arginyl residues in bovine myelin basic protein (MBP) on its susceptibility to digestion by cathepsin D has been studied. Using bovine component 1 (C-1) of MBP, the most unmodified of the components with all 18 arginyl residues intact, we have generated a number of citrullinated forms by treatment of the protein with purified peptidylarginine deiminase (PAD) in vitro. We obtained species containing 0-9.9 mol of citrulline/mol of MBP. These various species were digested with cathepsin D, a metalloproteinase which cleaves proteins at Phe-Phe linkages. The rate of digestion compared to component 1 was only slightly affected when 2.7 or 3.8 mol of citrulline/mol of MBP was present. With 7.0 mol of citrulline/mol of MBP, a large increase in the rate of digestion occurred. No further increase was observed with 9.9 mol of citrulline/mol of MBP. The immunodominant peptide 43-88 (bovine sequence) was released slowly when 2.7 and 3.8 mol of citrulline/mol of MBP was present, but it was released rapidly when 7.0 mol of citrulline/mol of MBP was present. The dramatic change in digestion with 7.0 mol of citrulline/mol of MBP or more could be explained by a change in three-dimensional structure. Molecular dynamics simulation showed that increasing the number of citrullinyl residues above 7 mol/mol of MBP generated a more open structure, consistent with experimental observations in the literature. We conclude that PAD, which deiminates arginyl residues in proteins, decreases both the charge and compact structure of MBP. This structural change allows better access of the Phe-Phe linkages to cathepsin D. This scheme represents an effective way of generating the immunodominant peptide which sensitizes T-cells for the autoimmune response in demyelinating disease.

Colorectal cancer is the second leading killer cancer worldwide and presently the most common cancer among males in Singapore. The study aimed to detect changes of protein profiles associated with the process of colorectal tumorigenesis to identify specific protein markers for early colorectal cancer detection and diagnosis or as potential therapeutic targets. Seven pairs of colorectal cancer tissues and adjacent normal mucosa were examined by two-dimensional gel electrophoresis at basic pH range (pH 7-10). Intensity changes of 34 spots were detected with statistical significance. 16 of the 34 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels revealed a significantly enhanced glycolytic pathway (Warburg effect), a decreased gluconeogenesis, a suppressed glucuronic acid pathway, and an impaired tricarboxylic acid cycle. Observed changes in protein abundance were verified by two-dimensional DIGE. These changes reveal an underlying mechanism of colorectal tumorigenesis in which the roles of impaired tricarboxylic acid cycle and the Warburg effect may be critical.

A comparison of extracellular proteins of virulent and avirulent Edwardsiella tarda strains revealed several major, virulent-strain-specific proteins. Proteomic analysis identified two of the proteins in the virulent strain PPD130/91 as flagellin and SseB, which are virulence factors in bacterial pathogens. PCR amplification and DNA sequencing confirmed the presence of the genes that encode these proteins. Our results clearly demonstrated the potency of the proteomic approach in identifying virulence factors.Edwardsiella tarda is a gram-negative enteric bacterial pathogen of both animals (6) and humans (14). Fish mortality caused by E. tarda infection (28) led to severe losses in the aquaculture industry. The bacterium's virulence features include the ability to invade epithelial cells (16,19), resistance to phagocytic killing (26), and production of enzymes such as hemolysins (5, 15) and chondroitinase (17). Functional genomic analysis is essential for a better understanding of the pathogenesis of E. tarda infections. The proteomic approach, which complements genomic research, has been employed to study the role of iron in regulating the pathophysiology of Mycobacterium tuberculosis (29) and the effect of environmental cues on gene expression within Salmonella pathogenicity island 2 (SPI2) in Salmonella enterica serovar Typhimurium (7). It was thus adopted here for the first time to compare and identify the secreted virulence factors of E. tarda.Defining virulence. Fourteen E. tarda strains were used for comparative proteomic analysis. The median 50% lethal doses (LD 50 s) of these strains were determined by using naïve blue gourami, Trichogaster trichopterus (Pallas), as described previously (19). Of the 14 strains used, 6 were described as virulent (LD 50 of Ͻ10 6.5 ) and 8 were described as avirulent (LD 50 of Ͼ10 7.0 ) ( Table 1). Culture conditions. Tryptic soy agar (TSA) medium (Difco) is used for growing E. tarda cultures in our laboratory. Brain heart infusion agar medium (Difco), which has also been used by others to culture E. tarda (27), was included for comparison. We first checked the extracellular protein (ECP) profiles of the two representative strains (virulent PPD130/91 and avirulent PPD125/87) cultured on TSA and brain heart infusion agar for 24 and 48 h at 25°C. ECP was prepared as described by Leung and Stevenson (18), with slight modifications. The final filtered ECP obtained was desalted and concentrated with a Millipore Biomax-5K column, and the protein concentration was determined by the Bio-Rad protein assay. One-dimensional (1D) polyacrylamide gel electrophoresis (PAGE) of the ECP was then performed according to standard procedures (22). Since the ECP production pattern was not affected by varying the culture media (data not shown), TSA was chosen for subsequent E. tarda cultures, and incubations of 24 instead of 48 h were used to avoid possible protein degradation due to a prolonged incubation period.1D PAGE profile analysis. Once the culture conditions had been fixed, the ECP profil...

Deimination of myelin basic protein (MBP) has been implicated in the chemical pathogenesis of multiple sclerosis (MS). Degradation of bovine MBP by cathepsin D, a myelin-associated protease, was increased when 6 arginyl residues were deiminated and became very rapid when all 18 arginyl residues were deiminated. Since MBP contains a number of modifications, including methylation, phosphorylation, etc., we studied the effect of methylation, an irreversible modification, to determine how this modification affected deimination. Methylation of Arg 106 in bovine MBP (Arg 107 in human), a naturally occurring modification of MBP, has been shown to affect the deimination of arginyl residues in the present study. Since fractionation of MBP into unmethylated, monomethylated, and dimethylated species cannot be done readily on a preparative scale, mass spectrometry with the Q-TOF instrument resolved these species readily since each differed from the other by 14 atomic mass units (amu). Examination of five different hMBP samples, two from normal brain and three from MS brain, revealed that increased deimination of arginyl residues correlated with a decreased methylation of Arg 107 (human sequence). To study this process in vitro, bovine MBP (bMBP) was used. Component 1 (C-1) is the most cationic of the MBP "charge isomers" and the most unmodified, in which all arginyl residues are intact. It was deiminated to various extents with purified bovine brain peptidylarginine deiminase, generating a number of species containing 0-13.7 mol of citrulline/mol of bMBP. Mass spectrometry of each of these species permitted us to determine the influence of methylation of Arg 106 (bovine sequence) on deimination by this enzyme. We found that bMBP with unmethylated arginine was deiminated at a rate of 0.081 mol of citrulline/min, with monomethylarginine, 0.068 mol of citrulline/min, and with dimethylarginine, 0.036 mol of citrulline/min. We suggest that the methylated arginyl residue becomes sequestered in the hydrophobic beta-sheet structure and disrupts the three-dimensional structure of the protein so that other arginyl residues are less accessible to peptidylarginine deiminase.

The amino acid sequences of the two major antifreeze polypeptides (AFP) from the shorthorn sculpin have been determined using an automatic protein sequencer and enzymic digestion. These two polypeptides, SS-3 and SS-8, consist of 33 and 45 amino acid residues respectively. The N-terminal methionyl residue is blocked in both the polypeptides. When aligned for maximum structural similarity these two AFP are 80% homologous, and there appears a deletion of 12 amino acid residues at the N-terminal portion of SS-3. Like the winter flounder AFP, both the sculpin AFP also contain the 11-amino-acid repeat sequences. The secondary structure of the sculpin AFP is mainly a-helical as deduced from circular dichroic spectral data. The helical content of SS-8 is high (73%), while that of SS-3 is moderate (about 45%). The latter exhibits a relatively weak antifreeze activity. Removal of the blocked N-terminal residue in SS-8 did not alter the helical content significantly but did reduce the antifreeze activity. Helical contents of proteolytically generated fragments of AFP are much lower, and they are devoid of activity. The a-helix in the SS-8 component is seen to be amphiphilic in character. The relevance of this feature to the mechanism of the antifreeze action is briefly discussed.To avoid freezing, many species ofmarine fishes inhabiting the Newfoundland and Labrador coastal waters produce antifreeze polypeptides (AFP) in the winter months [I -31. These species include the winter flounder [4-61, shorthorn sculpin [7], sea raven [8] and ocean pout [9]. Isolation and characterization of these AFP have indicated three distinct types of polypeptides based on the differences in amino acid composition and secondary structure [4-91. The AFP from flounder and sculpin comprise one such class.The AFP from the winter flounder, Pseudopleuronectus americunus can be fractionated into a t least seven active components by reverse-phase high-performance liquid chromatography (HPLC) [6]. The two major components, each with a relative molecular mass of about 3300, are closely related. Both consist of eight or nine different amino acids of which alanine accounts for 60% of the residues [4-61. The amino acid sequence of these two components have been elucidated [lo -121. The 37-residue-long polypeptide chain in each component has been found to contain three repeating sequences of 11 amino acid residues, Thr-(X)2-Y-(X)7-, where X represents a non-polar amino acid (mainly alanine) and Y is a polar amino acid [lo-121. The AFP from the shorthorn sculpin, Myoxocephulus scorpius, are similar to those of the winter flounder with respect to the abundance of alanine in their amino acid compositions and their high cr-helical contents [7, 131. However, they contain 12 different amino acids, and have slightly larger molecular masses withCorrespondence to

Cell membrane proteome analysis is limited by inherent membrane hydrophobicity. Conventional membrane protein extraction techniques use detergents, chaotropes and organic acids that require sample clean-up or pH adjustment, and are associated with significant sample loss. We extracted membrane proteins from red blood cells (RBCs) using methanol (MeOH), trifluoroethanol (TFE) and urea, and identified membrane proteins using 2-D LC coupled with MALDI-TOF/TOF-MS. We show that organic solvents MeOH- and TFE-based methods have membrane protein analysis efficiencies comparable to urea, and are complementary for the recovery of both hydrophilic and hydrophobic peptides. The mean grand average of hydropathicity (GRAVY) value of identified peptides from the TFE-based method (-0.107) was significantly higher than that of the MeOH-based method (-0.465) (p<0.001). Sequential and adjunctive use of the organic solvents MeOH and TFE increases the number of proteins identified, and the confidence of their identification. We show that this strategy is effective for shotgun membrane proteome analysis.

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