Tài liệu Báo cáo khóa học: Heterogeneity of homologously expressed Hypocrea jecorina (Trichoderma

Heterogeneity of homologously expressed Hypocrea jecorina

(Trichoderma reesei ) Cel7B catalytic module

Torny Eriksson1,*, Ingeborg Stals2,*, Anna Colle´ n1

, Folke Tjerneld1

, Marc Claeyssens2

, Henrik Sta˚ lbrand1

and Harry Brumer3

1

Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden; 2

Laboratory for

Biochemistry, Department of Biochemistry, Physiology and Microbiology, Ghent University, Belgium; 3

Department of Biotechnology,

Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, Sweden

The catalytic module of Hypocrea jecorina (previously

Trichoderma reesei) Cel7B was homologously expressed by

transformation of strain QM9414. Post-translational modi￾fications in purified Cel7B preparations were analysed by

enzymatic digestions, high performance chromatography,

mass spectrometry and site-directed mutagenesis. Of the

five potential sites found in the wild-type enzyme, only

Asn56 and Asn182 were found to be N-glycosylated.

GlcNAc2Man5 was identified as the predominant N-glycan,

although lesser amounts of GlcNAc2Man7 and glycans

carrying a mannophosphodiester bond were also detected.

Repartition of neutral and charged glycan structures over

the two glycosylation sites mainly accounts for the observed

microheterogeneity of the protein. However, partial deami￾dation of Asn259 and a partially occupied O-glycosylation

site give rise to further complexity in enzyme preparations.

Keywords: protein glycosylation; O-glycan;N-glycan;Tricho￾derma reesei; cellulase.

The filamentous fungus Hypocrea jecorina (previously

Trichoderma reesei [1]) produces several extracellular cellu￾lases, which cooperate in the degradation of paracrystalline

cellulose. The five known endoglucanases, including Cel7B,

generally act by hydrolysing the b(1fi4) glucan chains

internally [2,3], whereas the two cellobiohydrolases (Cel6A

and Cel7A) release cellobiose from the nonreducing and

reducing chain ends, respectively [4,5]. b-Glucosidase even￾tually hydrolyses this cellobiose to glucose which is taken up

by the fungal hyphae. All but one of the Hypocrea jecorina

cellulases share a similar modular structure comprised

of a catalytic module connected to a carbohydrate-binding

module (CBM) by a flexiblelinker peptide.The 3D structures

of the catalytic modules of Cel7A (formerly cellobiohydro￾lase I, CBH I [6]) and Cel7B (formerly endoglucanase I,

EG I) both exhibit a similar overall fold but are different in

their active site topologies; the former has a tunnel-shaped

active site whereas the latter possesses an open cleft [7,8]. This

reflects their different specificity, i.e. exo vs. endo activity [8].

The catalytic modules of fungal glycoside hydrolases are

often glycosylated on asparagine residues in the consensus

sequence Asn-Xaa-(Ser/Thr), where Xaa is not Pro [9]. This

post-translational modification is thought to affect protein

secretion and enzyme stability [10,11]. Of the structures

studied so far, most fungal N-glycans contain the mamma￾lian-type core structure (Man3GlcNAc2) [12]. However, the

occurrence of a single N-acetyl glucosamine on Cel7A from

H. jecorina strains ALKO2877 and QM9414 [13,14], indi￾cates glycosylation may be processed differently in some

cases. The N-glycosylation of both Cel7A and Cel7B,

isolated from different strains and grown under different

conditions, has been studied by several groups, but dispar￾ate and inconclusive results have been published [8,13–18].

The HypocreajecorinaCel7B catalytic module (Swiss-Prot

number P07981) possesses five potential N-glycosylation

sites. Single N-acetyl glucosamine (GlcNAc) residues have

been observed by X-ray crystallography on Asn56 and

Asn182 of Cel7B produced in the H. jecorina strain QM9414

[8]. In a later study, this enzyme was suggested to carry only

one high mannose N-glycan, some forms of which carried

mannophosphodiester linkages [16]. O-Mannosylation was

also indicated by this study, but the sites of attachment of

this and the N-glycan were not determined. Recently, Cel7B

from the H. jecorina strain Rut-C30 was shown to bear a

single GlcNAc on Asn56, while Asn182 was occupied with

higher-order glycans, primarily GlcNAc2Hex8 [18].

In the present study, we describe the glycoform analysis

of the catalytic module of H. jecorina Cel7B homologously

expressed in a QM9414 decendent strain using a range of

experimental techniques. Detailed analysis using mass

spectrometry and high-performance chromatography indi￾cated that two of the five potential N-glycosylation sites of

Correspondence to H. Brumer, Department of Biotechnology,

Royal Institute of Technology (KTH), AlbaNova

University Centre, S-106 91 Stockholm, Sweden.

Fax: + 46 85537 8468, Tel.: + 46 85537 8367,

E-mail: [email protected]

Abbreviations: Endo H, Streptomyces plicatus endoglycosidase H;

CID MS/MS, collision-induced dissociation tandem mass spectro￾metry; HPAEC-PAD, high-performance anion-exchange chromato￾graphy with pulsed amperometric detection; PAG-IEF,

polyacrylamide gel isoelectric focusing.

Enzyme: endoglycosidase H (EC 3.2.1.96).

*Note: These authors contributed equally to this work.

Note: A website is available at http://www.biotech.kth.se/

woodbiotechnology/

(Received 18 November 2003, revised 16 January 2004,

accepted 6 February 2004)

Eur. J. Biochem. 271, 1266–1276 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04031.x