PCR cloning and heterologous expression of cDNA encoding a peptidyl-Lys metalloendopeptidase precursor of Grifola frondosa.

Saito, Tamio; Dohmae, Naoshi; Tsujimoto, Masafumi; Takio, Koji

doi:10.2323/jgam.48.287

Cited by 7 publications

(9 citation statements)

References 22 publications

(24 reference statements)

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“…Lys-N is a zinc metalloendopeptidase that selectively cleaves the amide bond N-terminal of lysine residues. This enzyme, extracted from the fruiting body of basidomycete Grifola frondosa, has been sequenced, cloned, and crystallized 13-15. It is active in a pH range of 6.0 to 10.5, with an optimal pH reported as either 8.516 or 9.5 17.…”

Section: Introductionmentioning

confidence: 99%

Proteomic Analyses Using Grifola frondosa Metalloendoprotease Lys-N

et al. 2009

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Proteomic analysis typically has been performed using proteins digested with trypsin because of the excellent fragmentation patterns they produce in collision induced dissociation (CID). For analyses in which high protein coverage is desirable, such as global monitoring of post-translational modifications, additional sequences can be seen using parallel digestion with a second enzyme. We have benchmarked a relatively obscure basidomycete-derived zinc metalloendopeptidase, Lys-N, that selectively cleaves the amide bond N-terminal of lysine residues. We have found that Lys-N digestion yields peptides with easily assigned CID spectra. Using a mixture of purified proteins as well as a complex yeast lysate, we have shown that Lys-N efficiently digests all proteins at the predicted sites of cleavage. Shotgun proteomics analyses of Lys-N digests of both the standard mixture and yeast lysate yielded peptide and protein identification numbers that were generally comparable to trypsin digestion, whereas the combination data from Lys-N and trypsin digestion substantially enhanced protein coverage. During CID fragmentation, the additional amino terminal basicity enhanced b-ion intensity which was reflected in long b-ion tags that were particularly pronounced during CID in a quadrupole. Finally, immonium ion peaks produced from Lys-N digested peptides originate from the carboxy terminus in contrast to tryptic peptides where immonium ions originate from the amino terminus.

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Section: Introductionmentioning

confidence: 99%

Proteomic Analyses Using Grifola frondosa Metalloendoprotease Lys-N

et al. 2009

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“…We report cloning and expression of rAm-LysN in Escherichia coli strain BL21 (DE3) and P. pastoris. The homologous Gf-LysN has previously been expressed in P. pastoris, but in low quantities of 100 ng from an undisclosed volume (9). Our expression system in P. pastoris yields approx.…”

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confidence: 99%

Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

et al. 2015

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A method to express, purify and modify the PeptidylLys metallopeptidase (LysN) of Armillaria mellea in Pichia pastoris was developed to enable functional studies of the protease. Based on prior work, we propose a mechanism of action of LysN. Catalytic residues were investigated by site-directed mutagenesis. As anticipated, these mutations resulted in significantly reduced catalytic rates. Additionally, based on molecular modelling eleven mutants were designed to have altered substrate specificity. The S 1 0 binding pocket of LysN is quite narrow and lined with negative charge to specifically accommodate lysine. To allow for arginine specificity in S 1 0 , it was proposed to extend the S 1 0 binding pocket by mutagenesis, however the resulting mutant did not show any activity with arginine in P 1 0 . Two mutants, A101D and T105D, showed increased specificity towards arginine in subsites S 2 0 S 4 0 compared to the wild type protease. We speculate that the increased specificity to result from the additional negative charge which attract and interact with positively charged residues better than the wild type.Keywords: enzyme kinetics/enzyme mechanism/ metalloprotease/molecular modelling/site-directed mutagenesis.Abbreviations: Am-LysN, Armillaria mellea peptidyl-Lys metallopeptidase; DsbA, disulfideoxidoreductase; FRET, Fo¨rster resonance energy transfer; GAP, glyceraldehyde-3-phosphatedehydrogenase; IPTG, isopropylb-D-1-thiogalactopyranoside; MOE; Molecular Operation Environment (CCG); MOPS, 3-(N-morpholine)propanesulfonic acid; MS, mass spectrometry; PCR, polymerase chain reaction; RMSD, root-mean-square deviation; SDSPAGE, sodiumdodecylsulfatepolyacrylamide gel electrophoresis.In this article, we use Schechter and Berger nomenclature (1).Peptidyl-Lys metallopeptidase (LysN) from Armillaria mellea is an aspzincin metalloprotease, which cleaves in front of lysines (2). Hence, it is unusual by having its substrate specificity at the C-terminal side of the scissile bond (/K) (3). The protease is predominantly used as a tool in proteomic studies, where it serves as an alternative or complement to trypsin. The advantage of LysN is that it generates fragments which have a positive charge at the N-terminus, yielding mass spectrometry (MS) fragmentation with clear b-ion ladders (4,5). This feature can be used to facilitate de novo peptide mapping. Recently we profiled the substrate specificity of AmLysN and found it to be strictly lysine specific at S 1 0 , and having decreasing specificity when moving away from the scissile bond (2, 6).We also reported a homology model of Am-LysN based on the crystal structure of Gf-LysN (7). The two structures had a RMSD of 0.001 on backbone residues and 0.239 on the side chain residues (2). In this model Am-LysN has a narrow, and relatively short (57 amino acid) binding cleft. Central to the hydrolysis of peptides, is the zinc ion and the zinc binding motif 'HExxH + D'. Fushimi et al. showed that the aspartic acid D132 is necessary to coordinate zinc binding of the homologous protein ...

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“…Bacterial and fungal aspzincins have signal peptides targeting them for secretion (Saito et al 2002;Schwenteit et al 2013a); therefore, I investigated whether this secretion signature is conserved in the …”

Section: Sequence Characteristics Analysesmentioning

confidence: 99%

“…Characterised bacterial and fungal aspzincins have signal peptides and are secreted as a toxin (Saito et al 2002;Schwenteit et al 2013a). Forty-three of the 90 A. queenslandica aspzincins contain a…”

Section: Characteristics Of a Queenslandica Aspzincins: Secretion Tmentioning

confidence: 99%

“…Bacterial and fungal aspzincins with characterised proteolytic activity are synthesised as 37-41 kDa inactive precursors composed of a signal peptide, a propeptide, and the catalytic domain (McAuley et al 2001;Saito et al 2002;Doi et al 2004;Schwenteit et al 2013a). While the specific function of the aspzincin propeptide is not clear, as proposed for the…”

Section: The Aspzincins Of a Queenslandica Probably Still Have At Lementioning

confidence: 99%

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Horizontal gene transfer in the sponge Amphimedon queenslandica

Higgie¹

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Horizontal gene transfer (HGT) is the nonsexual transfer of genetic sequence across species boundaries.Historically, HGT has been assumed largely irrelevant to animal evolution, though widely recognised as an important evolutionary force in bacteria. From the recent boom in whole genome sequencing, many cases have emerged strongly supporting the occurrence of HGT in a wide range of animals.However, the extent, nature and mechanisms of HGT in animals remain poorly understood. Here, I explore these uncertainties using 576 HGTs previously reported in the genome of the demosponge Amphimedon queenslandica.The HGTs derive from bacterial, plant and fungal sources, contain a broad range of domain types, and many are differentially expressed throughout development. Some domains are highly enriched; phylogenetic analyses of the two largest groups, the Aspzincin_M35 and the PNP_UDP_1 domain groups, suggest that each results from one or few transfer events followed by post-transfer duplication.Their differential expression through development, and the conservation of domains and duplicates, together suggest that many of the HGT-derived genes are functioning in A. queenslandica. The largest group consists of aspzincins, a metallopeptidase found in bacteria and fungi, but not typically in animals.I detected aspzincins in representatives of all four of the sponge classes, suggesting that the original sponge aspzincin was transferred after sponges diverged from their last common ancestor with the Eumetazoa, but before the contemporary sponge classes emerged. In A. queenslandica, the aspzincins may have been co-opted for multiple functions, since 54 of the 90 fit into one of four ontogenetic expression profiles, each of which is putatively co-expressed with different suites of native genes.Based on secretion signals and the conservation of key catalytic residues, I propose that proteolytic activity is maintained in at least one of the aspzincin roles in A. queenslandica.Mobile elements are capable of genomic excision, movement and integration, they enable HGT in bacteria, and some animal HGTs are genomically close to eukaryotic transposable elements (TEs); as such, mobile elements are speculated as possible players in the mechanisms of interkingdom and interdomain HGT to animals. In A. queenslandica, the overall repeats densities around predicted Together, these results offer novel insight on HGT in A. queenslandica and demonstrate that HGT has a varied nature in this animal with an extensive impact, partially due to post-transfer duplications. Further, this work offers insights on possible mechanisms that led to the HGT derived genes of this sponge. iv Declaration by authorThis thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis.

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PCR cloning and heterologous expression of cDNA encoding a peptidyl-Lys metalloendopeptidase precursor of Grifola frondosa.

Cited by 7 publications

References 22 publications

Proteomic Analyses Using Grifola frondosa Metalloendoprotease Lys-N

Proteomic Analyses Using Grifola frondosa Metalloendoprotease Lys-N

Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

Horizontal gene transfer in the sponge Amphimedon queenslandica

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