Abstract

From the albumin gland of the snail Cepaea hortensis we isolated and characterized a new N-acetyl-D-galactosamine/N-acetyl-D-glucosamine (GalNAc/GlcNAc) specific lectin (CHA-II) which was purified by a combination of affinity chromatography on GalNAc-agarose and gel filtration. The purified native lectin was found to be a multimeric protein, as revealed by SDS–PAGE and MALDI-TOF analysis. In SDS–PAGE the denatured and reduced lectin showed two bands of molecular masses with 17 and 15.5 kDa which reacted equally with anti-CHA-II rabbit antiserum. The lectin was O- and N-glycosylated with [(Gal)₂-Man]₂-Man-GlcNAc-GlcNAc-Asn as a probable structure for the oligosaccharide. Isoelectric focusing revealed a heterogeneous protein of at least four bands around pH 8.7. Tryptic peptides of CHA-II were N-terminally sequenced and highly degenerated gene specific oligonucleotide primers (GSPs) had been constructed. Using total RNA isolated from albumin glands, cDNAs were produced by the running race technique. Specific PCR fragments were obtained by PCR using GSPs, the universal primer and 5′- or 3′-RACE-cDNAs. The amplified fragments were cloned into the vector pDrive and were sequenced. The resulting total cDNA sequence consisted of 496 base pairs including an open reading frame of 360 base pairs which encoded a protein of 120 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to comprise 99 amino acid residues with a calculated molecular weight of 11,239 Da. The PCR fragment encoding the mature protein was cloned into the vector pQE30 and expressed in E. coli. Recombinant CHA-II lectin was produced as inclusion bodies and extracted by 6 M guanidine hydrochloride. After refolding, the recombinant CHA-II agglutinated specifically human red blood cells of groups A and AB. In immunodiffusion experiments using rabbit antiserum raised against the native lectin, the protein showed a precipitation line of identity with the native lectin.

Keywords: Lectin; GaINAc; GlcNAc; Snail; Purification; Cloning; Sequencing

Article Outline

1. Introduction

2. Materials and methods

2.1. Isolation and characterization of the GalNAc/GlcNAc specific lectin CHA-II

2.1.1. Extraction, purification and characterization of the lectin

2.1.2. Carbohydrate analysis and MALDI-TOF analysis

2.1.3. Chemical deglycosylation

2.1.4. Protein determination and haemagglutination assay

2.1.5. Determination of amino acid sequence

2.1.6. Antisera production and Western blotting

2.2. Cloning the gene gagg-II of the GalNAc/GlcNAc agglutinating lectin CHA-II

2.2.1. RNA preparation and first strand cDNA synthesis

2.2.2. Preparation of gene-specific PCR fragments

2.2.3. Construction of the expression vector

2.2.4. Isolation and characterization of the recombinant lectin rCHA-II

3. Results

3.1. Characterization of the native GalNAc/GlcNAc-specific lectin (CHA-II)

3.2. Carbohydrate analysis of CHA-II

3.3. Amino acid sequence analysis

3.4. MALDI-TOF analysis and comparison with SDS–PAGE

3.5. Identification of the CHA-II encoding gene gagg-IIs

3.6. Biological characterization of native and recombinant lectin CHA-II

4. Discussion

Acknowledgements

References