Post-translational Modification of Thrombospondin Type-1 Repeats in ADAMTS-like 1/Punctin-1 by C-Mannosylation of Tryptophan

Protein C-mannosylation is the attachment of α-mannopyranose to tryptophan via a C-C linkage. This post-translational modification typically occurs within the sequence motif WXXW, which is frequently present in thrombospondin type-1 repeats (TSRs). TSRs are especially numerous in and a defining feature of the ADAMTS superfamily. We investigated the presence and functional significance of C-mannosylation of ADAMTS-like 1/punctin-1, which contains four TSRs (two with predicted C-mannosylation sites), using mass spectrometry, metabolic labeling, site-directed mutagenesis, and expression in C-mannosylation-defective Chinese hamster ovary cell variants. Analysis of tryptic fragments of recombinant human punctin-1 by mass spectrometry identified a peptide derived from TSR1 containing the ³⁶WDAWGPWSECSRTC⁴⁹ sequence of interest modified with two mannose residues and a Glc-Fuc disaccharide (O-fucosylation). Tandem mass spectrometry (MS/MS) and MS/MS/MS analysis demonstrated the characteristic cross-ring cleavage of C-mannose and identified the modified residues as Trp³⁹ and Trp⁴². C-Mannosylation of TSR1 of the related protease ADAMTS5 was also identified. Metabolic labeling of CHO-K1 cells or Lec35.1 cells demonstrated incorporation of d-[2,6-³H]mannose in secreted punctin-1 from CHO-K1 cells but not Lec35.1 cells. Quantitation of punctin-1 secretion in Lec35.1 cells versus CHO-K1 cells suggested decreased secretion in Lec35.1 cells. Replacement of mannosylated Trp residues in TSR1 with either Ala or Phe affected punctin secretion efficiency. These data demonstrate that TSR1 from punctin-1 carries C-mannosylation in close proximity to O-linked fucose. Together, these modifications appear to provide a quality control mechanism for punctin-1 secretion.The ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type-1 repeats) superfamily (1) consists of 19 secreted metalloproteases (ADAMTS proteases) and six ADAMTS-like proteins in humans. ADAMTS-like proteins closely resemble the ancillary domains of ADAMTS proteases and like them have a conserved modular organization containing one or more thrombospondin type-1 repeats (TSRs)² (2–5). TSRs are modules of ∼50 amino acids having a characteristic six-cysteine signature. The prototypic ADAMTSL, ADAMTSL1, also referred to as punctin-1 because of its punctate distribution in the substratum of transfected cells, is a 525-residue glycoprotein containing four TSRs (4). A longer punctin-1 variant arising from alternative splicing, containing 13 TSRs and homologous to ADAMTSL3, is predicted by the human genome sequencing project ({"type":"entrez-nucleotide","attrs":{"text":"NM_001040272","term_id":"334688819","term_text":"NM_001040272"}}NM_001040272) but has not yet been physically cloned and expressed. The function of ADAMTSL1/punctin-1 is unknown. Recently, ADAMTSL2 and ADAMTSL4 mutations were identified in the genetic disorders geleophysic dysplasia (6) and recessive isolated ectopia lentis, respectively (2). In genome-wide analysis, the ADAMTSL3 locus has been associated with variation in human height (7). Thus, in addition to known genetic disorders caused by ADAMTS mutations (8, 9), ADAMTSL family members are now also implicated in human disease. Among the ADAMTS proteases, ADAMTS5 and ADAMTS4 are strongly associated with cartilage destruction in arthritis (10–12).Like most secreted proteins, the ADAMTS superfamily members undergo post-translational modification and are predicted to contain N-linked oligosaccharides. In addition, TSRs of ADAMTS superfamily members, by virtue of high sequence similarity to the corresponding motifs in thrombospondin 1 and properdin, are predicted to contain two uncommon types of glycosylation. Specifically, TSRs often contain the sequence motifs W⁰XXW⁺³ and C¹X_2–3(S/T)C²XXG, which are consensus sites for protein C-mannosylation of the W⁰ residue and O-fucosylation (of Ser/Thr) respectively, in close proximity to each other (13, 14). In recently published work, it was shown that ADAMTSL1 and ADAMTS13 are modified by O-fucosylation, the covalent attachment to Ser or Thr residues of fucose or a fucose-glucose disaccharide (15, 16). Punctin-1 contains consensus sequences for O-fucosylation in all four of its TSRs, but the presence of the glycans was previously only confirmed on TSR2, -3, and -4 (16). Addition of O-fucose is mediated by protein O-fucosyltransferase 2 (POFUT2), which is a distinct transferase from that which catalyzes addition of O-linked fucose to epidermal growth factor-like repeats (POFUT1) (17, 18). A β3-glucosyltransferase subsequently adds glucose to the 3′-OH of the fucose (19, 20). It was further demonstrated that O-fucosylation, which occurs after completion of TSR folding, was rate-limiting for secretion of punctin-1 and ADAMTS13 (15, 16). This role was inferred from the following two experimental observations. 1) Expression of wild-type punctin-1 and ADAMTS13 in Lec13 cells, which do not fucosylate proteins, led to their decreased secretion (15, 16). 2) Mutation of the modified Ser or Thr residues greatly reduced secretion of punctin-1 and ADAMTS13 (15, 16).Protein C-mannosylation is the attachment of an α-mannopyranosyl residue to the indole C-2 of tryptophan via a C-C linkage (14, 21). Unlike O-fucosylation, it can utilize protein primary structure rather than tertiary structure as the determinant, i.e. mannose is added to unfolded polypeptides or unstructured synthetic peptides (22). C-Mannosylation uses dolichyl-phosphate mannose (Dol-P-Man) as the precursor and appears to be enzyme-catalyzed within the endoplasmic reticulum (23), but the responsible mannosyltransferase has not yet been identified. A variety of mammalian cell lines can perform this modification (24). Proteins known to be C-mannosylated include human RNase 2, interleukin 12, the mucins MUC5AC and MUC5B, and several proteins containing TSRs, such as thrombospondin-1, F-spondin, and components of complement (C6 and C7) and properdin (13, 21, 25–27).Krieg et al. (22) proposed a model in which the C-mannosyltransferase bound directly to the WXXW⁺³ motif, analogous to the Asn-X-(Thr/Ser) motif for N-glycosylation, and later analysis showed that both the Trp residues in the W⁰XXW⁺³XXX motif and the sole Trp residue in a (F/Y¹)XXW⁺³ motif could be modified (13). Based on meta-analysis of the C-mannosylation literature, Julenius (28) used a neural network approach to develop a prediction algorithm for protein C-mannosylation, termed NetCGlyc. This analysis suggested that Cys was an acceptable substitute for Trp at the +3 position (i.e. permitting C-mannosylation of W⁰ in a W⁰SSC motif). Julenius (28) reported a clear preference for small and/or polar residues (Ser, Ala, Gly, and Thr) at the +1 position, whereas Phe and Leu were not allowed. The NetCGlyc algorithm provides a useful guide for prediction of C-mannosylation sites, especially in the ADAMTS superfamily, which has a large number of TSRs (27). Here we specifically inquired whether the short form of punctin-1, the prototypic ADAMTSL, is modified by C-mannosylation, analyzed the role of Trp residues in the punctin TSRs, and investigated its possible functional significance in punctin-1 biosynthesis. By demonstrating that TSR1 of ADAMTS5 is also C-mannosylated, we extended the analysis to identify this unusual modification in an ADAMTS protease.

TABLE 1

Predicted C-mannosylation sites^a in the ADAMTS superfamilyOpen in a separate window^aThe full-length human reference ADAMTS sequences from GenBank™ were analyzed at the NetCGly 1.0 server for prediction of C-mannosylation sites. For prediction of O-fucosylation sites in the same peptide, the consensus sequence C¹X_2–3(S/T)C² XXG was utilized.^bThe sequence context in which the predicted modified Trp residue occurs is provided, and the residue with predicted modification is numbered. Ser/Thr residues predicted to be O-fucosylated based on the consensus sequence CXX(S/T)C are underlined.^cSequences containing predicted C-mannosylation sites that are not within TSRs are shown in italics.     

Synergy between Trehalose and Hsp104 for Thermotolerance in Saccharomyces Cerevisiae

We isolated a mutant strain unable to acquire heat shock resistance in stationary phase. Two mutations contributed to this phenotype. One mutation was at the TPS2locus, which encodes trehalose-6-phosphate phosphatase. The mutant fails to make trehalose and accumulates trehalose-6-phosphate. The other mutation was at the HSP104 locus. Gene disruptions showed that tps2 and hsp104 null mutants each produced moderate heat shock sensitivity in stationary phase cells. The two mutations were synergistic and the double mutant had little or no stationary phase-induced heat shock resistance. The same effect was seen in the tps1 (trehalose-6-phosphate synthase) hsp104 double mutant, suggesting that the extreme heat shock sensitivity was due mainly to a lack of trehalose rather than to the presence of trehalose-6-phosphate. However, accumulation of trehalose-6-phosphate did cause some phenotypes in the tps2 mutant, such as temperature sensitivity for growth. Finally, we isolated a high copy number suppressor of the temperature sensitivity of tps2, which we call PMU1, which reduced the levels of trehalose-6-phosphate in tps2 mutants. The encoded protein has a region homologous to the active site of phosphomutases.     

Phylogenetic analysis of the 90 kD heat shock family of protein sequences and an examination of the relationship among animals, plants, and fungi species

The heat shock protein (Hsp) sequences, because of their ubiquity and high degree of conservation, provide useful models for phylogenetic analysis. In this paper I have carried out a global alignment of all available sequences (a total of 31) for the 90-kD heat shock protein (Hsp90) family. The minimum amino acid identity that is seen between presently known Hsp90 homologs is about 40% over the entire length, indicating that it is a highly conserved protein. Based on the alignment, a number of signature sequences that either are distinctive of the Hsp90 family or that distinguish between the cytosolic and the endoplasmic reticular forms of Hsp90 have been identified. Detailed phylogenetic analyses based on Hsp90 sequences reported here strongly indicate that the cytosolic and the endoplasmic reticulum (ER) resident forms of Hsp90 constitute paralogous gene families which arose by a gene duplication event that took place very early in the evolution of eukaryotic cells. A minimum of two additional gene duplication events, which took place at a later time, are required to explain the presence of two different forms of Hsp90 that are found in fungi and vertebrate species. In a consensus neighbor-joining bootstrap tree based on Hsp90 sequences, plants and animals species grouped together 989 times of 1,000 (a highly significant score), indicating a closer relationship between them as compared to fungi. A closer affiliation of plant and animal species was also observed in the maximum-parsimony tree, although the relationship was not significantly supported by this method. A survey of the recent literature on this subject indicates that depending on the protein sequence and the methods of phylogenetic analysis, the animal species are indicated as closer relatives to either plants or fungi with significant statistical support for both topologies. Thus the relationship among the animal, plant, and fungi kingdoms remains an unresolved issue at the present time.      

Protein-based phylogenies support a chimeric origin for the eukaryotic genome

The phylogenetic position of the archaebacteria and the place of eukaryotes in the history of life remain a question of debate. Recent studies based on some protein-sequence data have obtained unusual phylogenies for these organisms. We therefore collected the protein sequences that were available with representatives from each of the major forms of life: the gram-negative bacteria, gram-positive bacteria, archaebacteria, and eukaryotes. Monophyletic, unrooted phylogenies based on these sequence data show that seven of 24 proteins yield a significant gram-positive-archaebacteria clade/gram-negative- eukaryotic clade. The phylogenies for these seven proteins cannot be explained by the traditional three-way split of the eukaryotes, archaebacteria, and eubacteria. Nine of the 24 proteins yield the traditional gram-positive-gram-negative clade/archaebacteria-eukaryotic clade. The remaining eight proteins give phylogenies that cannot be statistically distinguished. These results support the hypothesis of a chimeric origin for the eukaryotic cell nucleus formed from the fusion of an archaebacteria and a gram-negative bacteria.      

Orally bioavailable nonpeptide vitronectin receptor antagonists with efficacy in an osteoporosis model.

A new series of potent nonpeptide vitronectin receptor antagonists, based on a novel carbocyclic Gly-Asp mimetic, has been discovered. A representative of this series, SB 265123 (4), has 100% oral bioavailability in rats, and is orally active in vivo in the ovariectomized rat model of osteoporosis.     

Differential terminal fucosylation of N-linked glycans versus protein O-fucosylation in leukocyte adhesion deficiency type II (CDG IIc)

LAD II/CDG IIc is a rare autosomal recessive disease characterized by a decreased expression of fucosylated antigens on cell surfaces that results in leukocyte adhesion deficiency and severe neurological and developmental abnormalities. Its molecular basis has been identified as a defect in the transporter of GDP-l-fucose into the Golgi lumen, which reduces the availability of the substrate for fucosyltransferases. During metabolic radiolabeling experiments using [3H]fucose, LAD II fibroblasts incorporated significantly less radiolabel compared with control cells. However, fractionation and analysis of the different classes of glycans indicated that the decrease in [3H]fucose incorporation is not generalized and is mainly confined to terminal fucosylation of N-linked oligosaccharides. In contrast, the total levels of protein O-fucosylation, including that observed in Notch protein, were unaffected. This finding demonstrates that the decrease in GDP-l-fucose levels in the fibroblast Golgi caused by the LAD II defect does not impair bulk protein O-fucosylation, but severely affects the bulk addition of fucose as a terminal modification of N-linked glycans. These data suggest that the severe clinical abnormalities including neurological and developmental ones observed in at least some of the LAD II patients may be related to alteration in recognition systems involving terminal fucose modifications of N-glycans and not be due to a defective O-fucosylation of proteins such as Notch.     

Cyclo(l-Pro-d-Arg): a new antibacterial and antitumour diketopiperazine from Bacillus cereus associated with a rhabditid entomopathogenic

In continuation of our search for new antimicrobial secondary metabolites from Bacillus cereus associated with rhabditid entomopathogenic nematode, a new microbial diketopiperazine, cyclo(l-Pro-d-Arg), was isolated from the ethyl acetate extract of fermented modified nutrient broth. The chemical structures of the isolated compounds were identified based on their 1D, 2D NMR and high-resolution electrospray ionisation–mass spectroscopy data. Antibacterial activity of the compound was determined by minimum inhibitory concentration and disc diffusion method against medically important bacteria, and the compound was recorded to have significant antibacterial activity against test bacteria. The highest activity was recorded against Klebsiella pneumoniae (1 μg/mL). Cyclo(l-Pro-d-Arg) was recorded to have significant antitumor activity against HeLa cells (IC₅₀ value 50 μg/mL), and this compound was recorded to have no cytotoxicity against normal monkey kidney cells (VERO) up to 100 μg/mL). To the best of our knowledge, this is the first time that cyclo(l-Pro-d-Arg) has been isolated from a microbial natural source.     

Genetic elements associated with antimicrobial resistance in enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) from Brazil

Background  

We recently observed an association of resistance with a certain enteropathogenic Escherichia coli (EPEC) serotypes and identified a conjugative plasmid, similar to plasmid pED208, that was conserved among archival O111:H2/NM and O119:H2 strains of diverse geographical origin. In this study, we sought to determine the prevalence and distribution of this plasmid among a collection of EPEC isolates from Brazil, as well as to study the susceptibilities of these isolates to antimicrobial agents.     

Immunohistochemical localization of TLR2 and CD14 in gingival tissue of healthy individuals and patients with chronic periodontitis

We used immunohistochemistry to quantify and compare the expression of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14) in gingival tissues of both healthy individuals and patients with chronic periodontitis. We also correlated the expression of TLR2 and CD14 with the histological grades of chronic periodontitis. We examined 30 gingival specimens from chronic periodontitis patients and 10 from healthy individuals. Tissues from both groups were immunostained with antibodies against TLR2 and CD14. TLR2 and CD14 were expressed by endothelial cells, fibroblasts, lymphocytes and plasma cells. The immunohistochemical expression of TLR2 and CD14 was significantly greater in inflammatory cells of the chronic periodontitis group than in healthy individuals. Expression of these molecules was greater in the inflammatory cells of connective tissue adjacent to pocket epithelium in both groups. The expression of TLR2 and CD14 was greatest in the periodontitis group that was classified as severe grade, followed by moderate and mild grades, which suggests a role of TLR2 and CD14 in the pathogenesis of chronic periodontitis. The positive correlation of TLR2 and CD14 expression levels with the severity grades of chronic periodontitis suggests that they are correlated also with disease severity; therefore, they may be useful for predicting disease progression. Our findings are consistent with the possibility that CD14 acts as a co-receptor for TLR2.     

Synapsins Contain O-Linked N-Acetylglucosamine

The neuron-specific synaptic vesicle-associated phosphoproteins synapsin I and synapsin II were shown to contain terminal N-acetylglucosamine (GlcNAc) residues as determined by specific labeling with bovine galactosyltransferase and UDP-[3H]galactose. The beta-elimination of galactosyltransferase radiolabeled synapsin I and subsequent analysis of released saccharide on high-voltage paper electrophoresis confirmed the presence of monosaccharidic GlcNAc moieties in O-linkage to the protein. Partial cleavage of synapsin I by collagenase, 2-nitro-5-thiocyanobenzoic acid, and Staphylococcus aureus V8 protease suggests that at least three glycosylation sites exist along the molecule. Taken together these data present the first evidence that a neuron-specific protein contains O-glycosidically bound GlcNAc.