Raquel Montesino

The 1,4-β-d-glucan cellobiohydrolases from Phanerochaete chrysosporium. I. A system of synergistically acting enzymes homologous to Trichoderma reesei

A physico-chemical and structural characterization of three 1,4-beta-D-glucan cellobiohydrolases (EC. 3.2.1.91), isolated from a culture filtrate of the white-rot fungus Phanerochaete chrysosporium, reveals that the cellulolytic enzyme secretion pattern and thus the general degradation strategy for P. chrysosporium is similar to that of Trichoderma reesei. Partial sequence data show that two of the isolated enzymes, i.e., CBHI, pI 3.82 and CBH62, pI 4.85, are homologous with CBHI and EGI from T. reesei; while, the third, i.e., CBH50, pI 4.87, is homologous to T. reesei CBHII. Limited proteolysis with papain cleaved each of the three enzymes into two domains: a core protein which retained full catalytic activity against low molecular weight substrates and a peptide fragment corresponding to the cellulose binding domain, in striking similarity to the structural organization of T. reesei. CBHI and CBH62 have their binding domain located at the C-terminus, whereas in CBH50 it is located at the N-terminus. It is evident that synergistically acting cellobiohydrolases is a general requirement for efficient hydrolysis of crystalline cellulose by cellulolytic fungi.

Cloning and characterization of a cDNA encoding a cellobiose dehydrogenase from the white rot fungus Phanerochaete chrysosporium

The cDNA of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium has been cloned and sequenced. The 5′ end was obtained by PCR amplification. The cDNA contains 2310 translated bases excluding the poly(A) tail. The deduced mature protein contains 770 amino acid residues and is preceded by a 18 residue long signal peptide. The regions of the amino acid sequence corresponding to the heme and FAD domains of CDH were identified as well as the nucleotide‐binding motif, the disulfide pairing and a methionine residue chelating the heme iron. No homologous sequences were found for the heme domain, however, the FAD domain appears to be distantly related to the GMC oxidoreductase family.

Characterization of protein glycoforms with N‐linked neutral and phosphorylated oligosaccharides: studies on the glycosylation of endoglucanase 1 (Cel7B) from Trichoderma reesei

Using anion‐exchange chromatography the catalyticdomain of endoglucanase 1 (Cel7B) from Trichoderma reesei was resolved in multiple fractions with different isoelectric points, presumably related to different glycoforms of the enzyme. The protein fractions were analysed using lectins and electrospray MS. Isolated N‐glycans were analysed by fluorophore‐assisted carbohydrate electrophoresis and amine‐adsorption HPLC. The results show that this particular preparation contained at least 14 different glycoforms. The major isoform contained only one GlcNAc, presumably N‐linked, and one mannose, most probably O‐linked to serine/threonine at a separate site. Except for a small population containing Man5GlcNAc2+1–2 Man, the rest of the protein had negatively charged phosphate‐containing N‐glycans. All glycoforms contained at least one O‐linked mannose residue. The increased negative charge of the protein, introduced by oligosaccharide phosphorylation, is the most probable reason for the different isoelectric points and the occurrence of multiple peaks during purification.

Influence of culture conditions on the N-glycosylation of a monoclonal antibody specific for recombinant hepatitis B surface antigen

MAbs (monoclonal antibodies) are becoming increasingly important as diagnostic tools for pharmaceutical biotechnology, and hence it is crucial that they are produced under controlled conditions to assure their consistency and reproducibility, not only in terms of protein sequence and bioactivity, but also in terms of post‐translational modifications, e.g. for N‐glycosylation. Hybridoma CB.Hep‐1, which secretes an IgG2b mAb, was cultured in vivo in ascites and in vitro in static‐flask, spinner‐flask, dialysis‐membrane and perfusion systems using protein‐free, low‐serum‐containing medium (1% foetal‐calf serum) and high‐serum‐containing medium (8% foetal‐calf serum). These CB.Hep‐1 mAbs were fully characterized, and insignificant differences in the affinity constant were observed. Glycosylation profiling was performed by labelling the N‐glycans released by peptide N‐glycosidase F with either of the fluorophore tags 8‐aminonaphthalene‐1,3,6‐trisulphonic acid and 4‐aminobenzoic acid. The mAb produced in vivo showed two major biantennary‐complex‐type N‐glycans: monogalactosylated, core‐fucosylated and agalactosylated, core‐fucosylated. The mAbs produced in vitro in static flasks and spinner flasks were not significantly influenced by the serum content in the culture media and showed a higher degree of N‐glycan galactosylation compared with those produced in mouse‐ascites, hollow‐fibre and membrane systems. The monogalactosylated, core‐fucosylated structure was the most abundant N‐glycan except for those produced in ascites and hollow fibres, where the agalactosylated, core‐fucosylated glycoform was the major specie. MAbs produced in high‐cellular‐yield systems displayed greater galactosylation heterogeneity influenced by changes in culture media.

Cellobiose quinone oxidoreductase from the white rot fungus Phanerochaete chrysosporium is produced by intracellular proteolysis of cellobiose dehydrogenase

The fungus Phanerochaete chrysosporium was grown in a 10-l automatic fermenter using cellobiose as carbon source to monitor the induction of cellobiose dehydrogenase (CDH) and cellobiose quinone oxidoreductase (CBQ) enzymes, and to search for tentative cbq and cdh genes and their transcriptional products. After 24 h of induction, CDH was detected in the culture supernatant and a protein was recognized by a specific anti-CDH polyclonal antibody in the sonicated biomass. Northern blot experiments performed with several fungal RNA samples showed, after 24 h of induction, only one single species of an mRNA transcript corresponding in size to the cdh gene (2.5 kb) The relative amount of this transcript decreased as a function of time. Southern blot experiments done with genomic DNA and database search in the recently available genome information also ruled out the presence in this strain of a separate cbq gene distinct from the cdh gene. Taken together, these results demonstrated that CBQ originates from the cdh gene. Furthermore, it is not produced by differential splicing but by a posttranslational, predominantly intracellular, proteolytic cleavage.

Structural characterization of N-linked oligosaccharides on monoclonal antibody Nimotuzumab through process development.

Nimotuzumab (TheraCIM, CIMAher, h-R3, humanized anti-EGF-R antibody), monoclonal antibody (mAb) manufactured at the Center of Molecular Immunology (Havana, Cuba) is currently being tested in several clinical trials. Nimotuzumab has a single N-glycosylation site in the Fc-CH2 fragment but no N-glycosylation site in the Fab region. The current study reports the full characterization of the mAb N-glycosylation and the consistency observed in several production batches from a perfusion mode culturing system that lasted between 68 and 150 days. It confirms that the N-glycan structures of Nimotuzumab expressed in the NS0 murine myeloma cell line are of the murine type. They consist mainly of fucosylated G0, G1 and G2 oligosaccharides, which are normally found in the CH2 region of IgG. Other minor species found were high mannose and sialylated structures. A small portion of the glycans were sialylated (∼12%) and the only type of sialic acid detected was N-glycolyl-sialic acid, α2,6-linked to Gal. No Galα1-3Gal moieties were detected.

N-glycosylation pattern of E2 glycoprotein from classical swine fever virus.

The extracellular domain of E2 glycoprotein outer surface of the classical swine fever virus was expressed in epithelial kidney pig cells. The N-glycosylation determined by combination of Normal Phase-HPLC, Weak Anion Exchange-HPLC, exoglycosidase digestions and Mass Spectrometry revealed a complex mixture of neutral and monosialylated multiantennary N-glycans with variable number of alpha1-3-Gal-Gal antennae terminals. The most abundant neutral N-glycan has a composition of Hex(7)HexNAc(4)dHex(1), Negative ion ESI-MS/MS confirmed the presence of the alpha1-3-Gal-Gal motif on each arm of the fucosylated biantennary N-glycan. The most abundant monosialylated glycan was Hex(6)HexNAc(4)dHex(1)Neu(5)Ac(1), with the sialic acid linked to the terminal beta1-4-Gal-GlcNAc. Sialic acid on the antenna capping position was predominantly of the N-acetyl form.

Thermal stress treatment does not affect the stability and protective capacity of goat milk derived E2-marker vaccine formulation against CSFV

Classical swine fever virus produces a huge mortality in infected herds during recurrent outbreaks, predominantly in tropical and subtropical areas. In this scenario, it is common that cold-chain related issues affect the efficacy of virus attenuated-derived vaccines, which are frequently used in eradication programs. In the present work, the stability and protective capacity of a recombinant vaccine preparation, based on goat milk derived E2 glycoprotein extracellular domain, were both analyzed after incubation at 4 degrees C or 37 degrees C for 1 week. Differences in the viscosity and in the homodimeric form of the antigen were observed after comparing physicochemical properties of stressed and not stressed vaccine formulations. However, these differences did not affect the immunogenicity and protective capacity of such preparations. Noticeably, pigs immunized with the E2-based vaccine subjected to thermal stress became totally protected from the viral infection, after a challenge with 10(5) PLD(50) of a high virulent classical swine fever strain. This result supports the practical value of this vaccine preparation mostly for those regions in which cold-chain related failures tend to affect the protective capability of conventional virus attenuated vaccines.

The N-glycosylation of classical swine fever virus E2 glycoprotein extracellular domain expressed in the milk of goat.

Classical swine fever virus (CSFV) outer surface E2 glycoprotein represents an important target to induce protective immunization during infection but the influence of N-glycosylation pattern in antigenicity is yet unclear. In the present work, the N-glycosylation of the E2-CSFV extracellular domain expressed in goat milk was determined. Enzymatic N-glycans releasing, 2-aminobenzamide (2AB) labeling, weak anion-exchange and normal-phase HPLC combined with exoglycosidase digestions and mass spectrometry of 2AB-labeled and unlabeled N-glycans showed a heterogenic population of oligomannoside, hybrid and complex-type structures. The detection of two Man(8)GlcNAc(2) isomers indicates an alternative active pathway in addition to the classical endoplasmic reticulum processing. N-acetyl or N-glycolyl monosialylated species predominate over neutral complex-type N-glycans. Asn207 site-specific micro-heterogeneity of the E2 most relevant antigenic and virulence site was determined by HPLC-mass spectrometry of glycopeptides. The differences in N-glycosylation with respect to the native E2 may not disturb the main antigenic domains when expressed in goat milk.