Getting Started

Andrea Rodríguez-Martín, Raquel Acosta, Félix Núñez, Mª José Benitoa_{, Miguel A.} Asensio*

Higiene y Seguridad Alimentaria, Facultad de Veterinaria. Universidad de Extremadura, Avda. de la Universidad, s/n. 10071- Cáceres, Spain.

a _{Nutrición y Bromatología, Escuela de Ingenierías Agrarias, Universidad de Extremadura, Carretera de}

Cáceres s/n, 06071- Badajoz, Spain

* Corresponding author: Telephone (+34) 927 257 125; fax: (+34) 927 257 110. E-mail: [email protected]

ABSTRACT

The protein PgChP is a new chitosanase with antifungal activity produced by Penicillium chrysogenum AS51D. The gene encoding the chitosanase PgChP has been sequenced. A DNA fragment of the gene encoding pgchp was obtained by PCR using degenerate primers based on two known amino acid sequences of the purified PgChP. cDNA was prepared for RACE-PCR from total RNA of P. chrysogenum by RT-PCR. Full-length cDNA and complete genomic DNA of pgchp were obtained using primers designed from 5’ and 3’ – ends. Comparing the cDNA and genomic DNA, four introns of 60, 53, 59, and 59 bp were observed. Full-length cDNA has 915 bp, corresponding to an open reading frame encoding a protein of 304 amino acid residues. The first 21 amino acids matched a signal peptide. The predicted mass of the translated nucleotidic sequence was 32 kDa and the predicted isoelectric point was 4.8. The translated sequence of pgchp revealed that the protein had two carboxylic amino acids (Glu and Asp) described as essential for the catalytic function of fungal chitosanases. Two N-glycosylation consensus sequences were observed in the translated pgchp gene. The putative amino acid sequence showed high homology with fungal chitosanases.

KEY WORDS: antifungal activity, glycosylation, N-linked oligosaccharides, dry- fermented foods.

INTRODUCTION

P. chrysogenum AS51D was isolated from meat products in previous studies (Acosta et al., 2009a). This mould produces the protein PgChP, selected for its ability of inhibiting the

growth of some toxigenic moulds. In previous works, different de novo amino acid sequences obtained from PgChP showed significant similarities with the respective sequences of seven fungal chitosanases, belonging to the GH-75 family of fungal chitosanases (Rodríguez-Martín et al., 2009). Chitosanases (E.C. 3.2.1.132) are produced by a large number of microorganisms including bacteria and fungi. Numerous bacterial chitosanases are able to degrade exogenously added chitosan (Shimosaka et al., 2000; Yoon et al., 2002; Lee et al., 2006; Zhang et al., 2007). On the contrary, fungal chitosanases have been reported from only a limited number of strains and their physiological roles are unclear (Fenton and Eveleigh, 1981; Zhang et al., 2000). The synthesis of some chitosanases can be induced by adding different substrates such as glucosamine (Zhang et al., 2001) and chitosan (Zhu et al., 2007).

Analysis of the molecular weight of PgChP by ESI-MS revealed six isoforms differing from each other by approximately 162 Da, the mass of one residue of mannose. These evidences indicated that the chitosanase PgChP was a glycoprotein with different degrees of glycosylation. Various deglycosylation treatments revealed the presence of N-linked oligosaccharides in PgChP (Rodríguez-Martín et al., 2009).

The aim of this paper was to study the genetic sequence encoding the chitosanase PgChP. This sequence will allow studying the existence of introns, the complete putative amino acid sequence, and glycosylation consensus sequences.

MATERIALS AND METHODS Microbial strain

P. chrysogenum AS51D was isolated from dry-cured ham and belongs to the fungal collection of Food Hygiene, University of Extremadura.

DNA isolation

P. chrysogenum AS51D was grown in malt extract broth made with 2% (w/v) glucose, 2% (w/v) malt extract, and 0.1% (w/v) peptone, pH 4.5 at 25ºC under continuous shaking, for 5 days. The mycelium was obtained by filtering the culture through paper Whatman nº2. Two grams of mycelium were broken with mortar and pestle after freezing by adding liquid nitrogen. Fungal lysis was performed by homogenizing the obtained powder in 4 ml 0.05 M Tris-HCl, pH 8; 0.005 M ethylenediamine tetraacetic acid (EDTA); 0.05 M NaCl

(TES) buffer and 1% (w/v) sodium dodecyl sulfate (SDS). Next, 200 µl proteinase K (20 µg/µl) were added, and the mix was incubated at 60ºC for 35 min, cooled on ice and extracted with phenol-chloroform-isoamyl alcohol (25:24:1). The suspension was centrifuged at 3000×g for 2 min at 4ºC. The upper phase containing DNA was transferred to a fresh tube and precipitated by adding 3 M sodium acetate pH 5.2 to a final concentration of 10% (w/v) and two volumes of cold ethanol. After centrifugation, the pellet was cleaned with 70% (v/v) ethanol, centrifuged again, resuspended in sterile water, and treated with 50 µl RNase (10 µg/µl) at 37ºC for 60 min. Finally, DNA was extracted with phenol-chloroform-isoamyl alcohol, and precipitated again as indicated above, resuspended in water, and stored at -70ºC. Quantity and quality of purified DNA was determined spectrophotometrically in a Biophotometer (Eppendorf AG, Hamburg, Germany).

Genomic DNA amplification

Polymerase chain reactions were performed with genomic DNA (100 ng) as template in 50 µl reaction mixtures containing 50 pmol each of forward and reverse degenerated primers, 0.5 mM each of deoxyribonucleotide triphosphates, (dNTPs, Roche, Madrid, Spain), 0.1 vol of 10× PCR buffer (22.5 mM MgCl2, 500 mM Tris-HCl pH 9.2, 140 mM ammonium

sulfate), 1.8 mM MgCl2 and 2.5 units of pfu turbo polymerase (Stratagene, La Jolla, USA).

The degenerated primers pgchp-DPF1 and pgchp-DPR2A (Table 1) were designed from amino acid sequences previously obtained by mass spectrometry (Rodríguez-Martín et al., 2009). Additionally, for pgchp-DPR2A the contiguous residues conserved in fungal chitosanases (Shimosaka et al., 2005) were also included.

DNA amplification was carried out in a Px2 thermocycler (Thermo Scientific, Cramlington, UK). Three reactions were done, one with both forward and reverse primers, and two with either forward or reverse primers. The PCR program consisted of initial denaturation (94ºC for 5 min), 35 cycles of denaturation (94ºC for 1min), annealing (56- 60ºC range for 30 s, gradient 12), and extension (72ºC for 30 s), followed by a final extension at 72ºC for 4 min. PCR products were electrophoresed on 2% (w/v) agarose gels in 1× TAE buffer (40 mM Tris-acetate, 1 mM EDTA), pH 8 ussing a Sub-Cell GT apparatus (Bio-rad Laboratories, Madrid, Spain). Two different markers were used for agarose gels: 1kb DNA ladder (0.25-12 kb bands) (Promega, Hampshire, United Kingdom) and DNA size standard (0.05-2kb) (Bio-Rad). Then, products detected only in the reactions with both reverse and forward primers were gel-purified and cloned into the pCR2.1- TOPO vector (Invitrogen, Barcelona, Spain). One Shot TOP10 Chemically Competent E. coli cells (Invitrogen) were transformed with that vector. To confirm the insert in the vector, digestions with restriction enzyme EcoRI (Roche) were carried out. Finally, vectors were sequenced in the Institute of Biomedicine (CSIC, Valencia, Spain).

RNA isolation

P. chrysogenum AS51D was grown in malt extract broth, pH 4.5 at 25ºC under continuous shaking for 4 days. Chitosanase production was induced in a medium with glucosamine (Zhang et al., 2001) made with 0.3% (w/v) NaNO3, 0.05% (w/v) KCl, 0.05% (w/v)

MgSO4•7H2O, 0.01% (w/v) FeSO4•7H2O, and 2% (w/v) D(+)-glucosamine hydrochloride

(Sigma, Dorset, United Kingdom), dissolved in 25 mM phosphate buffer pH 5.8. Mould Table 1. Degenerated primers used to amplify the pgchp gene.

Primer names Amino acid sequences and designed primers*

pgchp-DPF1

N K P D G G P 5’-AAY AAR CCN GAY GGN GGN CC-3’

pgchp-DPR2A

M A R T C F P 3’-AC CGN GCN TGN ACR AAR GG-5’ * R for purine, Y for pirimidine, N for A, C, T, or G.

was cultured in glucosamine medium at 25ºC under continuous shaking for 17 h. Subsequently, mycelium was quickly frozen with liquid nitrogen and stored at -80ºC until RNA extraction. Frozen mycelium was broken with mortar and pestle after adding liquid nitrogen. RNA extraction was performed with RNeasy Plant Mini Kit (Qiagen, Crawley, UK) following the manufacturer’s instructions. Total extracted RNA was resuspended in 40 µl of RNase-free deionized water.

Rapid amplification of cDNA ends by Polymerase Chain Reaction (RACE-PCR) Amplification of 5’ and 3’ ends of the pgchp gene was carried out using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Saint Germain en Laye, France).

cDNA synthesis by reverse transcriptase-polymerase chain reaction (RT-PCR). Two separate populations of cDNA were synthesized according to the amplification kit instructions: 5’-RACE-Ready cDNA and 3’-RACE-Ready cDNA. After incorporating the SMART sequence into both the 5’- and 3’-RACE-Ready cDNA populations, RACE PCR reactions were performed using the Universal Primer A Mix (UPM), in conjunction with distinct gene-specific primers.

Gene-specific primers design. Two gene-specific primers (GSP) were designed for amplifying the 5’ and 3’- ends (5’-RACE GSP: CCATTGCAGATAACGGCACCAACG and 3’-RACE GSP: GTTCGACAAGGGTGCGGCATATG). Polymerase chain reactions were performed with 5’- or 3’-RACE-Ready cDNAs (50 ng) as template in 50 µl reaction

mixtures containing 1 mM of dNTPs (Roche), 0.5 µM of the 5’- or 3’-RACE GSP primers, 5 µl of Universal Primer A Mix (10×), 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2, and 2.5 units pfu turbo polymerase

(Stratagene). A program for touchdown PCR was carried out for 5’-end amplification: 5 cycles of 2 steps (94ºC for 30 s and 72ºC for 3 min), 5 cycles of 3 steps (94ºC for 30 s, 70ºC for 30 s and 72ºC for 3 min) and 27 cycles of 3 steps (94ºC for 30 s, 68ºC for 30 s and 72ºC for 3 min). The PCR program to obtain the 3’-end consisted of 30 cycles of denaturation (94ºC for 30 s), annealing (68ºC for 30 s), and extension (72ºC for 3 min). Finally, 5’- and 3’-RACE-PCR products were gel-purified and cloned into pCR2.1-TOPO vectors, which were used to transform One Shot TOP10 Chemically Competent E. coli (Invitrogen). Then plasmids were purified and sequenced. Digestions with restriction enzyme EcoRI (Roche) were performed to confirm the insert in the vector.

Full-length cDNA amplification. The full cDNA fragment of the gene pgchp was obtained by PCR with specific primers Fwd-pgchp (ATGATGACCTACAGCCGCTTAATCCC) and Rev-pgchp (TCAATCACTGGGGCATTTTCCAC) designed from 5’ and 3’ ends. PCR reaction mixtures at a final volume of 50 µl contained 5’-RACE-Ready cDNA (50 ng), 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase (Stratagene). The PCR program consisted of 30

cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were gel-purified and cloned into pCR2.1-TOPO vectors. These plasmids were used to transform One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). Then vectors were purified and sequenced. Digestions with restriction enzyme EcoRI (Roche) were performed to confirm the wanted insert in the vector.

Amplification of the whole genomic gene

The genomic gene sequence was amplified by PCR using Fwd-pgchp and Rev-pgchp primers. PCR reactions contained 100 ng of genomic DNA, 0.5 µM each of forward and reverse primers, 0.5 mM each of the dNTPs, 0.1 vol of 10× PCR buffer (10 mM Tris-HCl, pH 8.8, 50 mM KCl, 0.1% Triton X-100), 4 mM MgCl2 and 2.5 units pfu turbo polymerase

(Stratagene), and deionized water until a final volume of 50µl. The PCR program consisted of 30 cycles of denaturation (94ºC for 30 s), annealing (65ºC for 30 s), and extension (72ºC for 3 min). PCR products were gel-purified and cloned into pCR2.1-TOPO vectors. These plasmids were used to transform One Shot TOP10 Chemically Competent E. coli cells (Invitrogen). Then vectors were purified and sequenced. Alignment between gene sequences obtained from genomic DNA and cDNA was performed. Introns sequences were confirmed by GenScan software.

RESULTS

Partial amplification of pgchp gene using degenerated primers

The pgchp gene partial amplification from genomic DNA was done using two degenerated primers: pgchp-DPF1 and pgchp-DPR2A (Table 1). After different reactions, the optimal annealing temperature was 57.7ºC. PCR products obtained with these primers were run in

agarose gel. Just one band with high intensity was amplified in reactions with both forward and reverse primers (Figure 1). This band was cloned and sequenced.

After sequencing, the selected fragment was found to consist of 657 bp. The obtained sequence was translated and compared in Basic Local Alignment Search Tool (BLAST) with known sequences. A high degree of similarity with several fungal chitosanases was found, as it is discussed later. The common sequences included three introns described for genes encoding fungal chitosanases.

Sequence of the pgchp gene by RACE PCR

After induction with glucosamine, mRNA was extracted and used to obtain both 5’- and 3’-RACE-Ready cDNA populations by RT-PCR. Two gene specific primers were designed from the obtained genomic DNA sequence without introns to get 5’- and 3’-ends: 5’-RACE GSP (5’-CCATTGCAGATAACGGCACCAACG-3’) and 3’-RACE GSP (5’- GTTCGACAAGGGTGCGGCATATG-3’), respectively. After 5’ and 3’-RACE reactions, two bands with the expected size (Figure 2) were gel-purified from 5’- and 3’-RACE Figure 1. PCR products using forward DPF1 (F), both forward DPF1 and reverse DPR2A (F+R), and reverse DPR2A (R) primers. M: DNA standards. The arrow indicates the selected band.

PCRs, cloned, and sequenced. The cDNA ends of the gene 5’ and 3’ were 559 bp and 796 bp, respectively.

Next, two new primers were designed to obtain the full encoding cDNA (Fwd-pgchp: 5’-

ATGATGACCTACAGCCGCTTAATCCC-3’ and Rev-pgchp: 5’-

TCAATCACTGGGGCATTTTCCAC-3’). The full-length cDNA contained a 915 bp open reading frame encoding a protein of 304 amino acid residues (Figure 3) that included the known PgChP sequences.

Genomic DNA of pgchp gene, amplified using the same primers for cDNA, was 1146 bp length. Comparison of cDNA and genomic sequences revealed that pgchp gene had four introns with a length of 60, 53, 59, and 59 bp (Figure 3). These introns were confirmed by GenScan program.

Figure 2. cDNA fragments of 5’- and 3’-RACE PCR (A and B, respectively). M: DNA standards.

The open reading frame was translated to protein by ExPASy proteomics server at the Swiss Institute of Bioinformatics (http://www.expasy.org). Analysis of the signal peptidase cleavage site predicted by the SignalP program (Nielsen et al., 1997) suggested that the pre-sequence consisted of the first 21 amino acids (MMTYSRLIPFALFALFGTGLA).

5’-atgatgacctacagccgcttaatccccttcgctctctttgccttgtttggcacagggctc M M T Y S R L I P F A L F A L F G T G L gcgcaaacagtcgatggctccaaattcaacaagccagacggcggtccaccaggaagctac A Q T V D G S K F N K P D G G P P G S Y ttcgctgctggctcgtctattcccgtcgctgctttgcagagcgctgctgcaaaggctcgc F A A G S S I P V A A L Q S A A A K A R actcccgtgccagatgcaacctatcctattaatggcgataacggtgctaagaaagttacc T P V P D A T Y P I N G D N G A K K V T atccacagcgactgggctaagttcgacaaggtagatactctcactggtcggttaattgca I H S D W A K F D K << Intron 1 aggtccgcatgcttattaaatggatcatagggtgcggcatatgtttggattgcggatatg >> G A A Y V W I A D M gacgtcgactgcgacggcattgactacaagtgcaaggtacggtcactattgttgtctgtc D V D C D G I D Y K C K << Intron 2 tgagatgttattgctgacccgatgttcagggaaacccggatggtcagcaccaaaccaact >> G N P D G Q H Q T N tcggagctttggctgcgtatgaagtgccgttctttgtgattcccgacaggtttggaacca F G A L A A Y E V P F F V I P D R F G T agtacgcgaagcagcttcctggaaacaacgttggtgccgttatctggtatggtttcctac K Y A K Q L P G N N V G A V I C<< ctttgtaagatatctcagcggcgtcttgttctaaccgaaagctagcaatggaaagatgtt Intron 3 >> N G K M F ctacggaatttacggagactccgatggcgatacccctcaggtcatcggcgaggcctcatg Y G I Y G D S D G D T P Q V I G E A S W gttaatggcccggacctgcttccctaatgatgacttgaatggcaatagtggccatggtga L M A R T C F P N D D L N G N S G H G D tgttgatgtgacctgtaagtttgcagccttggcacatctagatccgtattctgtcaacta V D V T << Intron 4 ctaatctaaccagatatcctcttcactggcgacgactcggtgctccccagcagcgctctc >>Y I L F T G D D S V L P S S A L aacaagaattatgtcaccaacttcaccactctgcgctctatgggagacaaactcatgact N K N Y V T N F T T L R S M G D K L M T gctcttgcgaagaacctcaagttggtagatggaggtgatggaggttctccaacaactacg A L A K N L K L V D G G D G G S P T T T gctgggtccaaccctactagcgggtcttgtgagtgggagggacactgtgctggtgcgtct A G S N P T S G S C E W E G H C A G A S tgcaaagatgaaaatgattgctccgatcaactggtctgcaaaagtggaaaatgccccagt C K D E N D C S D Q L V C K S G K C P S gattga-3’ D -

Figure 3. Full-length DNA sequence of pgchp gene. The open reading frame has been translated to amino acids. Typical sequences of fungal introns are underlined.

DISCUSSION

RACE-PCR is a method widely used in the characterization of genes. The successful of this technique is highly depended on the amount of mRNA from the gene enconding the studied protein. Thus, the production of PgChP was induced by glucosamine, as it has been reported for other chitosanases (Zhang et al., 2001). After the mRNA extraction, both populations 5’- and 3’-RACE-Ready cDNAs were satisfactorily obtained by RT-PCR. Using gene specific primers designed from the sequence amplified with degenerated primers, the 5’- and 3’-ends were obtained by RACE-PCR.

When the complete sequence was obtained, a difference between the pgchp gene from genomic DNA (1146 bp) and from cDNA (915 bp) could be observed (Figure 3). Thus, the existence of four introns of 60, 53, 59, and 59 bp in the gene, which are absent in the cDNA, was confirmed by GenScan software. The small size of these introns compared to mammalian introns is a typical feature from fungal genes (Gurr et al., 1987). The 5’- and 3’-ends of the four introns are very similar (Figure 3) and coincide with the consensus splice sequences for fungal introns 5’-splice donor site (GT), the 3’-splice acceptor site (AG). In addition, three of them have the internal CTRAC sequence (Figure 3), typical of fungal introns (Wiesner et al., 1988).

The translated sequence of the gene pgchp encodes a chitosanase of 304 amino acids of the PgChP protein. Comparing the putative amino acid sequence of pgchp gene with seven fungal chitosanases, the homology was about 60 %. Several common sequences were found among all these proteins (Figure 4). Three conserved regions have been described in fungal chitosanases as important for catalytic function (Shimosaka et al., 2005). In two of these regions there are one aspartic and one glutamic acid residues, essentials for catalytic function of the chitosanase from F. solani. The predicted amino acid sequence of PgChP also shows these two residues (Figure 4). Carboxylic amino acids (Glu or Asp) have been identified as necessary residues for the catalytic function as proton donors in a large number of glycosyl hydrolases (Monzingo et al., 1996; Robertus et al., 1998). PgChP and the other seven fungal chitosanases show eleven common residues of aspartic acid as well as two of glutamic acid (Figure 4). Moreover, 10 cysteines of PgChP are found in all these fungal chitosanases (Figure 4). Cysteine residues are known to play an important role on the stabilization of the tertiary structure by the formation of disulfide bridges in fungal proteins (Nakaya et al., 1990; Lacadena et al., 1995; Marx et al., 1995; Lee et al., 1999).

PgChP --MMTYSRLIPFALFALFG-TGLAQTVDGSKFNKPDGGPPGSYFAAGSSIPVAALQSAAA 57 A.fumigatus MPSTTIIRQLAIS-LALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAA 58 N.fischeri MPSKTIIRQLAIS-VALCN-SALGQVVNGADYNKPNGGPPASFFAAASTMPVAALQAAAA 58 A.niger MAFKTTAG--LAF-LALAG-SVKAQSVDGSKYNSPSNGPPASYFAAATTLPVAALQSAAA 56 A.oryzae-1 MPIKSFASRLALS-LAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAA 58 A.oryzae-2 MPIKSFASRLALS-LAICG-TAMGQKVNGADYNKPDGGPPAKFFQASSSIPVAAIQAAAA 58 A.terreus MVFKKAAIGLTLP-FALFSSVALGQTVDGSDYDSPNGGPPGSYFAAASTMPVAALQAAAA 59 A.clavatus MIFKSSLSHAAAS-LLLLT-PALAQKVQGPEYNKPSAGPPASFFAAAPTMPVAALKSAVA 58 PgChP KARTPVPDATYPINGDNGAKKVTIHSDWAKFDKGAAYVWIADMDVDCDGIDYKC--- 111

A.fumigatus KATKVPSLATYPVSQDKGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC--- 112 N.fischeri KASKVPSLATYPVSQDSGAAKSTIHTDWASFSEGASISWVADMDVDCDGLNSGC--- 112 A.niger KASSVPSKATYPVNTDDDSPKSTIHSDWVKFNQGAALSWVADMDVDCDGIDYKC--- 110 A.oryzae-1 KASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC--- 110 A.oryzae-2 KASKVPSHATYPIGQ--GSTKSTIHSDWAGFSEGAAFSFIADMDVDCDGLNHGC--- 110 A.terreus KATKVPSYATYPVSQDDNAKKSTIHSDWASFSQGAAISWVADMDVDCDGIDSGCEHEIKT 119 A.clavatus RASVVPKNAAYPVNQD-GGPTATIHADWASLPTAAAYVYTADMDVDCDGLDHNC--- 111 PgChP -KGNPDGQHQTNFGALAAYEVPFFVIPDRFGTKYAKQLPGNNVGAVIC----NGKMFYGI 166 A.fumigatus -QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANSGALPGNNIAAVIC----NGKMFYGI 167 N.fischeri -QGNPDGQPQTNWGALSAYEVPFIVIPDKYLSANTGALPGNNIAAVIC----NGKMFYGI 167 A.niger -KGNGDGLPETNWGALSAYEVPWIVIPDQFLTANEDLLPGNNVAAVIC----NGKMYYGI 165 A.oryzae-1 -KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVIC----NGKMFYGI 165 A.oryzae-2 -KGNPDGQKETNWGALSAYEVPFIVIPQEFLDANKGTLKGNAVAAVICATSSNGKMFYGI 169 A.terreus SQGNPDGQDATNWGALAAYEVPFIVIPQKYLDHNGNALKGNNIAAVIC----NGKMFYGI 175 A.clavatus -KGNPDGQPDTNFGALAAYEVPFVVIPDRFATTYASALPGNNIVAVIC----DGKMFYGI 166

PgChP YGDSDGDTPQVIGEASWLMARTCFPNDDLNGNSGHGDVDVTYILFTGDDSVLPSSALNKN 226 A.fumigatus LGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKNAVLPSSALTKN 227 N.fischeri LGDSNGDSPQVTGEASWLMARTCFPNEGLNGNNGHTGVDVTYIVFTGKDAVLPSSALTKN 227 A.niger LGDSNGDDPEVTGEASWLMARTCFPDDDLNGAEGHAEADVTCKPYT---RSALNKN 218 A.oryzae-1 FGDSNGDSPQVTGEASWLMARTCFPKEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKN 225 A.oryzae-2 FGDSNGDSPQVTGEASWLMARTCFPEEDLNGNKGHTAADVTYIVFTGDKAVLPSSALNKN 229 A.terreus LGDANGDEPQVTGEASWLMARTCFPNEGLNGNKGHTAADVTYILFTGDESVLPSSALNEN 235 A.clavatus FGDTDGDHPQVIGEASWLMARTCFPNDNLNGDSGHVPADVTYIFFTGKDSVLPSSAVNKN 226 * *

PgChP YVTNFTTLRSMGDKLMTALAKNLKLVDGGDGG---SPT--TTAGSNPT--SGSC 273 A.fumigatus YITNFTTLRSMGDKLVNALVSNLGLSGTPPTK---TTTRVTTTTTKPT-SAASC 277 N.fischeri YITNFTTLRSMGDKLVNALASKLGLSGTPPTK---TTTLVTTTTTKPT-STASC 277 A.niger YITNFSTLRSMGDKLVGALASNLGLTSSA---SGSTATC 254 A.oryzae-1 YITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---PTTLTTTSIPEPTGGSGSC 276 A.oryzae-2 YITNFDTLRSMGDSLVGALAKNLNLGGGGGNP---PTTLTTTSIPEPTGGSGSC 280 A.terreus YITNFSTLRSMGDKLVNALASNLGLSGGSGGT---PTT--TTTATQPT-STGTC 283 A.clavatus YITDFTKLRSMGDSLVNAFASQLGISSGGGNGGGDGGHTTLHTTTATRTATAPT-STATC 285

PgChP EWEGHCA---GASCKDENDCSDQLVCKSGKCPSD--- 304

A.fumigatus SWAGHCL---GASCSSDDDCADALVCTAGKCSVDGAA-TCSWEGHCEGASCSSDDDCS 331 N.fischeri SWAGHCL---GASCSSNDDCADALVCAAGKCSVDGAA-TCSWEGHCEGVYS--- 324

A.niger SWQGHCE---GAICSTEDDCSDDLVCDSGKCSSPDED--- 288 A.oryzae-1 SWPGHCA---GATCSSNDDCSDDLTCQNGKCASDGSAETCSWEGHCKGATCSSNDDCS 331 A.oryzae-2 SWPGHCAGFKNKGATCSSNDDCSDDLACQNGKCASDGSAETCSWEGHCKGATCSSNDDCS 340 A.terreus SWAGHCE---GASCSTNDDCSDQLACKNGVCSTDGEV-VCSWEGHCEGATCSSNDDCS 337 A.clavatus DWEGHCL---GTACSNNGECSDPFGCINGFCNYDPTL-TCQWAGHCVGASCSSNDQCS 339 PgChP --- A.fumigatus DDLYCKSGSCTA---P--- 344 N.fischeri --- A.niger ---DGDDEDDEDEEENDEDDEDDDDEDDEDDE--- 317 A.oryzae-1 DELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--- 380 A.oryzae-2 DELACISGICSVDNGVETCEWEGHCEGASCSSHDDCDGNLACKNGKCSA--- 389 A.terreus DELYCAGGACTSA--- 350 A.clavatus DPFACIDGACAVDT-SLDCSRKGHCAGTTCLSDSDCSRPLSCILGVCANQSG 390

Figure 4. Alignment of PgChP with other fungal chitosanases. Common amino acids are denoted by background shaded letters. Asterisks indicate essential amino acid residues for catalytic activity of fungal chitosanases. Accession numbers in Genbank are XM_754126 (A. fumigatus), XM_001262949 (N. fischeri), XM_001390407 (A. niger), BAD08218 (A. oryzae-1), XP_001824257 (A. oryzae-2), XM_001209034 (A. terreus), XM_001274664 (A. clavatus).

Six glycosylated isoforms of PgChP with N-linked oligosaccharides have been reported (Rodríguez-Martín et al., 2009). In N-glycosylations, glycans are attached to the protein via N-acetylglucosamine (GlcNAc) to the amide group of asparagine within an Asn-X- Ser/Thr/Cys motif, where X is any amino acid apart from proline (Morelle et al., 2006). Two N-glycosylation consensus sequences were found in the translated pgchp gene: NFT (residues 230-232) and NDC (residues 288-290).

Glycosylation is the most common post-translational modification in proteins. Carbohydrates participate in important biological processes, including inter- and extracellular signalling, protein regulations and interactions, and molecular recognition. Glycans can determine the localization, activity and function of proteins, as well as influence on physicochemical characteristics of proteins such as folding, solubility, resistance to proteases or thermal stability (Geyer et al., 2006; Morelle et al., 2006). The potential role of the N-linked oligosaccharides of PgChP on intra- or extracellular