Porphyromonas gingivalis is an anaerobic, asaccharolytic Gram-negative rod associated with chronic periodontitis. We have undertaken a proteomic study of the outer membrane of P. gingivalis strain W50 using two-dimensional gel electrophoresis and peptide mass fingerprinting. Proteins were identified by reference to the pre-release genomic sequence of P. gingivalis available from The Institute for Genomic Research. Out of 39 proteins identified, five were TonB-linked outer membrane receptors, ten others were putative integral outer membrane proteins and four were putative lipoproteins. Pyroglutamate was found to be the N-terminal residue of seven of the proteins, and was predicted to be the N-terminal residue of 13 additional proteins. The RgpA, Kgp and HagA polyproteins were identified as fully processed domains in outer membranes prepared in the presence of proteinase inhibitors. Several domains were found to be C-terminally truncated 16-57 residues upstream from the N-terminus of the following domain, at a residue penultimate to a lysine. This pattern of C-terminal processing was not detected in a W50 strain isogenic mutant lacking the lysine-specific proteinase Kgp. Construction of another W50 isogenic mutant lacking the arginine-specific proteinases indicated that RgpB and/or RgpA were also involved in domain processing. The C-terminal adhesin of RgpA, designated RgpA27, together with RgpB and two newly identified proteins designated P27 and P59 were found to migrate on two-dimensional gels as vertical streaks at a molecular mass 13-42 kDa higher than that calculated from their gene sequences. The electrophoretic behaviour of these proteins, together with their immunoreactivity with a monoclonal antibody that recognizes lipopolysaccharide, is consistent with a modification that could anchor the proteins to the outer membrane.

The N-end rule pathway is conserved from bacteria to man and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing an N-degron are recognised by ClpS and degraded by ClpAP in an ATP-dependent manner. Here, we report the isolation of 23 ClpS-interacting proteins from E. coli. Our data show that at least one of these interacting proteins-putrescine aminotransferase (PATase)-is posttranslationally modified to generate a primary N-degron. Remarkably, the N-terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyltRNA-protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N-terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N-end rule pathway, through the ClpAPS-mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N-degron, which is required for substrate delivery to ClpA.

Caseinomacropeptide (CMP) is a heterogeneous C-terminal fragment (residues 106 to 169) of bovine milk -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 highperformance 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 -casein (residues 106 to 169) [-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 -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 -casein-A(138-158) and its nonphosphorylated counterpartcasein-A(138-158) were chemically synthesized and tested for antibacterial activity. The synthetic Ser(P) 149-casein-A(138-158) displayed growth-inhibitory activity against S. mutans (MIC, 59 g/ml [26 M]). The nonphosphorylated peptide, however, did not inhibit growth at the concentrations tested, indicating that phosphorylation is essential for activity.The caseins are the most abundant bovine milk proteins, and there are four major types: ␣ s1 -, ␣ s2 -, ␤-, and -casein (23). All four caseins are phosphorylated on specific seryl residues, and in addition, -casein is glycosylated (4). -Casein is hydrolyzed by the enzyme chymosin between Phe 105 and Met 106 , generating two polypeptides: a hydrophobic N-terminal para--casein polypeptide -casein (residues 1 to 105) [-casein(1-105)] and a hydrophilic phosphorylated and glycosylated C-terminal polypeptide -casein(106-169), known as the caseinomacropeptide (CMP). CMP is heterogeneous and contains all the posttranslational modification sites (glycosylation and phosphorylation) of -casein. Six potential glycosylation sites have been identified on CMP (18), and up to five different carbohydrate moieties may be attached at each site (20). Three genetic variants of CMP have also been identified, originating from the precursors -casein A, B, and E, with variants A and B being the most common in bovine milk (15).CMP and CMP-derived peptides have been reported to have a variety of biological activities, such as suppression of gastric secretions (28), depression of platelet aggregation (2), inhibition of influenza virus hemagglutination (8), inhibition of cholera toxin binding (9), and immunomodulating activities (14). CMP has also been shown to incorporate into salivary pellicle and inhibit the adherence of Streptococcus mutans, the oral...

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 β2-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.

[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.

The cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.

Several merozoite surface proteins are being assessed as potential components of a vaccine against Plasmodium falciparum, the cause of the most serious form of human malaria. One of these proteins, merozoite surface protein 2 (MSP2), is unusually hydrophilic and contains tandem sequence repeats, characteristics of intrinsically unstructured proteins. A range of physicochemical studies have confirmed that recombinant forms of MSP2 are largely unstructured. Both dimorphic types of MSP2 (3D7 and FC27) are equivalently extended in solution and form amyloid-like fibrils although with different kinetics and structural characteristics. These fibrils have a regular underlying β-sheet structure and both fibril types stain with Congo Red, but only the FC27 fibrils stain with Thioflavin T. 3D7 MSP2 fibrils seeded the growth of fibrils from 3D7 or FC27 MSP2 monomer indicating the involvement of a conserved region of MSP2 in fibril formation. Consistent with this, digestion of fibrils with proteinase K generated resistant peptides, which included the N-terminal conserved region of MSP2. A monoclonal antibody that reacted preferentially with monomeric recombinant MSP2 did not react with the antigen in situ on the merozoite surface. Glutaraldehyde cross-linking of infected erythrocytes generated MSP2 oligomers similar to those formed by polymeric recombinant MSP2. We conclude that MSP2 oligomers containing intermolecular β-strand interactions similar to those in amyloid fibrils may be a component of the fibrillar surface coat on P. falciparum merozoites.

The binding of CD4 and chemokine receptors to the gp120 attachment glycoprotein of human immunodeficiency virus triggers refolding of the associated gp41 fusion glycoprotein into a trimer of hairpins with a 6-helix bundle (6HB) core. These events lead to membrane fusion and viral entry. The envelope glycoprotein complex (Env) 4 of human immunodeficiency virus type 1 (HIV-1) comprises a trimer of receptor binding gp120 subunits in non-covalent association with a trimer of transmembrane gp41 subunits on the surface of infected cells and virions. Viral entry is initiated when gp120 binds to cell-surface CD4 molecules, inducing structural changes within the gp120 core domain, and leading to the formation of the binding site for the chemokine co-receptors, CCR5 and/or CXCR4 (1-4). gp120 comprises 5 variable loops (V1-V5) that exist outside the gp120 core; V3, and to a lesser degree V1 and V2, contribute to chemokine receptor binding specificity (5-7). The sequential binding of gp120 to CD4 and chemokine receptor triggers the refolding of gp41 into a trimer of hairpins, which mediates membrane fusion (8, 9). gp41 is a class I fusion glycoprotein, being structurally homologous to the fusion glycoproteins of other retroviruses, orthomyxoviruses, paramyxoviruses, filoviruses, and coronaviruses. The gp41 ectodomain is comprised of an N-terminal fusion peptide, connected through a flexible polar segment to a coiled coil-forming amphipathic ␣-helix (N-helix), a centrally located disulfide-bonded loop, a C-terminal amphipathic ␣-helix (C-helix), and a membrane-proximal tryptophan-rich region (MPR) (10 -16). The ectodomain is anchored to the viral envelope by a C-terminally located transmembrane domain (TMD), which precedes an ϳ150-residue cytoplasmic domain.The majority of the gp41 ectodomain appears to be buried by the gp120 trimer. This model for gp41 in the context of prefusion Env is based on the findings that the epitopes of monoclonal antibodies (mAbs) encompassing the fusion peptide and polar segment (residues 521-538), disulfide bonded region (579 -613), and C-helix (644 -663), are largely occluded in pre- * This work was supported by the National Health and Medical ResearchCouncil of Australia Grants 296200 and 345413, American Foundation for AIDS Research Grant 106610-36-RGNT, and Sidaction. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 4 The abbreviations used are: Env, envelope glycoprotein; HIV-1, human immunodeficiency virus type 1; N-helix, N-terminal coiled coil forming ␣-helix of gp41; C-helix, C-terminal ␣-helix of gp41; MPR, membrane proximal region; TMD, transmembrane domain; mAb, monoclonal antibody; sCD4, soluble CD4; DiO, 3,3Ј-dioctadecyloxacarbocyanine perchlorate; DiI, 1,1Ј-dioctadecyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine perchlorate; PBS, phosphate-buffered saline; MBP, maltose-binding protein; MALDI, matrixassisted la...