Complement factor C5 in Atlantic salmon (Salmo salar): characterization of cDNA, protein and glycosylation

Complement component 5 (C5) is an essential factor of the defensive complement system in all vertebrates. We report the characterization of C5 cDNA and protein from Atlantic salmon (Salmo salar), a teleost fish species of high importance in aquaculture. The C5 cDNA cloned from liver is 5079 nucleotides long, whose translation product has a molecular weight of 190 kDa, with the classical β-α orientation and motifs/sites for β-α cleavage (678RPKR681) and cleavage by C5 convertases (R758). Mass spectrometric analysis show a single N-linked, biantennary, complex glycan at N1125. Moreover, the N-linked glycan displays an unusual modification in the form of acetylated sialic acid residues. Three anti-C5 antisera produced in mice using purified C5 worked in immunohistochemical analyses of formalin fixed liver tissue. The purification method, whereby inactive and activated (C5b) forms were isolated, opens for interesting studies on the complement function in fish, including possible connection to stress, disease and glycosylation.publishedVersio

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium Streptococcus sanguinis

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, Streptococcus sanguinis. In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in S. sanguinis. We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, i.e. a methylatedN terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved byNMR,revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than bona fide Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that S. sanguinis Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines

Investigation of the genes coding for chaperonins in Chlorobium tepidum

A subgroup of the universally conserved molecular chaperones, the chaperonins assist other proteins in folding or refolding in an ATP- dependent manner. First discovered in E. coli the best described member is the GroEL (Hsp60) and GroES (Hsp10), which are often found together in an operon. The genes for these chaperonins are commonly regulated positive by an alternative sigma factor, as in E. coli or negatively by the CIRCE/ HrcA feedback regulatory system as in streptomyces. C. tepidum, a green sulphur bacterium, contains an operon consisting of groEL and groES, and a single groES-2 gene. In this work extensive analysis of the genetic sequences, in particular the regulatory upstream region was done. This information was used for structure modelling as well as to design primers for isolation and detection of genes by PCR. The isolated genes were subsequently ligated into a pET 102/ D TOPO® vector and expressed in transformed E. coli BL21 (DE) cells. All genes were expressed with a thioredoxin fusion protein in this work. To detect expression of groES, groEL and groES-2 genes in C. tepidum a Real Time reverse transcriptase PCR were done on RNA extracted from C. tepidum. However, this experiment encountered obstacles and were not finished due to lack of time

Large-scale intact glycopeptide identification by Mascot database search

Workflows capable of determining glycopeptides in large-scale are missing in the field of glycoproteomics. We present an approach for automated annotation of intact glycopeptide mass spectra. The steps in adopting the Mascot search engine for intact glycopeptide analysis included: (i) assigning one letter codes for monosaccharides, (ii) linearizing glycan sequences and (iii) preparing custom glycoprotein databases. Automated annotation of both N- and O-linked glycopeptides was proven using standard glycoproteins. In a large-scale study, a total of 257 glycoproteins containing 970 unique glycosylation sites and 3447 non-redundant N-linked glycopeptide variants were identified in 24 serum samples. Thus, a single tool was developed that collectively allows the (i) elucidation of N- and O-linked glycopeptide spectra, (ii) matching glycopeptides to known protein sequences, and (iii) high-throughput, batch-wise analysis of large-scale glycoproteomics data sets

Allelic polymorphisms in a glycosyltransferase gene shape glycan repertoire in the O-linked protein glycosylation system of Neisseria

Glycosylation of multiple proteins via O-linkage is well documented in bacterial species of Neisseria of import to human disease. Recent studies of protein glycosylation (pgl) gene distribution established that related protein glycosylation systems occur throughout the genus including nonpathogenic species. However, there are inconsistencies between pgl gene status and observed glycan structures. One of these relates to the widespread distribution of pglG, encoding a glycosyltransferase that in Neisseria elongata subsp. glycolytica is responsible for the addition of di-N-acetyl glucuronic acid at the third position of a tetrasaccharide. Despite pglG residing in strains of N. gonorrhoeae, N. meningitidis and N. lactamica, no glycan structures have been correlated with its presence in these backgrounds. Moreover, PglG function in N. elongata subsp. glycolytica minimally requires UDP-glucuronic acid (GlcNAcA), and yet N. gonorrhoeae, N. meningitidis and N. lactamica lack pglJ, the gene whose product is essential for UDP-GlcNAcA synthesis. We examined the functionality of pglG alleles from species spanning the Neisseria genus by genetic complementation in N. elongata subsp. glycolytica. The results indicate that select pglG alleles from N. meningitidis and N. lactamica are associated with incorporation of an N-acetyl-hexosamine at the third position and reveal the potential for an expanded glycan repertoire in those species. Similar experiments using pglG from N. gonorrhoeae failed to find any evidence of function suggesting that those alleles are missense pseudogenes. Taken together, the results are emblematic of how allelic polymorphisms can shape bacterial glycosyltransferase function and demonstrate that such alterations may be constrained to distinct phylogenetic lineages.publishedVersio

The Pediocin PA‑1 Accessory Protein Ensures Correct Disulfide Bond Formation in the Antimicrobial Peptide Pediocin PA‑1

Peptides, in contrast to proteins, are generally not large enough to form stable and well-defined three-dimensional structures. However, peptides are still able to form correct disulfide bonds. Using pediocin-like bacteriocins, we have examined how this may be achieved. Some pediocin-like bacteriocins, such as pediocin PA-1 and sakacin P­[N24C+44C], have four cysteines. There are three possible ways by which the four cysteines may combine to form two disulfide bonds, and the three variants are expected to be produced in approximately equal amounts if their formation is random. Pediocin PA-1 and sakacin P­[N24C+44C] with correct disulfide bonds were the main products when they were secreted by the pediocin PA-1 ABC transporter and accessory protein, but when they were secreted by the corresponding secretion machinery for sakacin A, a pediocin-like bacteriocin with one disulfide bond (two cysteines), peptides with all three possible disulfide bonds were produced in approximately equal amounts. All five cysteines in the pediocin PA-1 ABC transporter and the two cysteines (that form a CxxC motif) in the accessory protein were individually replaced with serines to examine their involvement in disulfide bond formation in pediocin PA-1. The Cys86Ser mutation in the accessory protein caused a 2-fold decrease in the amount of pediocin PA-1 with correct disulfide bonds, while the Cys83Ser mutation nearly abolished the production of pediocin PA-1 and resulted in the production of all three disufide bond variants in equal amounts. The Cys19Ser mutation in the ABC transporter completely abolished secretion of pediocin PA-1, suggesting that Cys19 is in the proteolytic active site and involved in cleaving the prebacteriocin. Replacing the other four cysteines in the ABC transporter with serines caused a slight reduction in the overall amount of secreted pediocin PA-1, but the relative amount with the correct disulfide bonds remained large. These results indicate that the pediocin PA-1 accessory protein has a chaperone-like activity in that it ensures the formation of the correct disulfide bond in pediocin PA-1

Allelic polymorphisms in a glycosyltransferase gene shape glycan repertoire in the O-linked protein glycosylation system of Neisseria

Glycosylation of multiple proteins via O-linkage is well documented in bacterial species of Neisseria of import to human disease. Recent studies of protein glycosylation (pgl) gene distribution established that related protein glycosylation systems occur throughout the genus including nonpathogenic species. However, there are inconsistencies between pgl gene status and observed glycan structures. One of these relates to the widespread distribution of pglG, encoding a glycosyltransferase that in Neisseria elongata subsp. glycolytica is responsible for the addition of di-N-acetyl glucuronic acid at the third position of a tetrasaccharide. Despite pglG residing in strains of N. gonorrhoeae, N. meningitidis and N. lactamica, no glycan structures have been correlated with its presence in these backgrounds. Moreover, PglG function in N. elongata subsp. glycolytica minimally requires UDP-glucuronic acid (GlcNAcA), and yet N. gonorrhoeae, N. meningitidis and N. lactamica lack pglJ, the gene whose product is essential for UDP-GlcNAcA synthesis. We examined the functionality of pglG alleles from species spanning the Neisseria genus by genetic complementation in N. elongata subsp. glycolytica. The results indicate that select pglG alleles from N. meningitidis and N. lactamica are associated with incorporation of an N-acetyl-hexosamine at the third position and reveal the potential for an expanded glycan repertoire in those species. Similar experiments using pglG from N. gonorrhoeae failed to find any evidence of function suggesting that those alleles are missense pseudogenes. Taken together, the results are emblematic of how allelic polymorphisms can shape bacterial glycosyltransferase function and demonstrate that such alterations may be constrained to distinct phylogenetic lineages

Type IV pilus assembly proficiency and dynamics influence pilin subunit phospho-form macro- and microheterogeneity in Neisseria gonorrhoeae

The PilE pilin subunit protein of the gonococcal Type IV pilus (Tfp) colonization factor undergoes multisite, covalent modification with the zwitterionic phospho-form modification phosphoethanolamine (PE). In a mutant lacking the pilin-like PilV protein however, PilE is modified with a mixture of PE and phosphocholine (PC). Moreover, intrastrain variation of PilE PC modification levels have been observed in backgrounds that constitutively express PptA (the protein phospho-form transferase A) required for both PE and PC modification. The molecular basis underlying phospho-form microheterogeneity in these instances remains poorly defined. Here, we examined the effects of mutations at numerous loci that disrupt or perturb Tfp assembly and observed that these mutants phenocopy the pilV mutant vis a vis phospho-form modification status. Thus, PC modification appears to be directly or indirectly responsive to the efficacy of pilin subunit interactions. Despite the complexity of contributing factors identified here, the data favor a model in which increased retention in the inner membrane may act as a key signal in altering phospho-form modification. These results also provide an alternative explanation for the variation in PilE PC levels observed previously and that has been assumed to be due to phase variation of pptA. Moreover, mass spectrometry revealed evidence for mono- and di-methylated forms of PE attached to PilE in mutants deficient in pilus assembly, directly implicating a methyltransferase-based pathway for PC synthesis in N. gonorrhoeae

Complement factor C5 in Atlantic salmon (Salmo salar): characterization of cDNA, protein and glycosylation

Complement component 5 (C5) is an essential factor of the defensive complement system in all vertebrates. We report the characterization of C5 cDNA and protein from Atlantic salmon (Salmo salar), a teleost fish species of high importance in aquaculture. The C5 cDNA cloned from liver is 5079 nucleotides long, whose translation product has a molecular weight of 190 kDa, with the classical β-α orientation and motifs/sites for β-α cleavage (678RPKR681) and cleavage by C5 convertases (R758). Mass spectrometric analysis show a single N-linked, biantennary, complex glycan at N1125. Moreover, the N-linked glycan displays an unusual modification in the form of acetylated sialic acid residues. Three anti-C5 antisera produced in mice using purified C5 worked in immunohistochemical analyses of formalin fixed liver tissue. The purification method, whereby inactive and activated (C5b) forms were isolated, opens for interesting studies on the complement function in fish, including possible connection to stress, disease and glycosylation