The Characterisation And Application Of Mannose-Binding Lectin Isolated From The Seeds Of Artocarpus Integer (Champedak)

Author

FAIZAH BINTI AHMAD

Date

2001

Keyword

Artocarpus integer, 'Champedak' , mannose-binding lectin, glycopeptides

Abstract

Champedak (Artocarpus integer) lectin-M is a lectin previously described with high specificity and affinity for the core-mannosyl residues of the N-linked oligosaccharides of glycoproteins. In the present study, a similar lectin was isolated and purified from the seeds of champedak cultivar CHI4. Our data demonstrated that the purified lectin was comparable with the earlier reported mannose-binding lectin-M that was isolated and purified from the seeds of champedak cultivar CH30. The lectin demonstrated a single band upon reacting with normal human serum in a double diffusion study and a 16.8-kDa non-reducing band in an SDS-PAGE experiment. The interaction of our champedak seed lectin-M with human serum glycoproteins that were resolved by 2-dimensional (2-D) gel electrophoresis was also studied. The lectin demonstrated strong interaction with haptoglobin ß chain, orosomucoid, α1-antitrypsin,α2-HS glycoprotein, transferrin, hemopexin, aiB-glycoprotein and the heavy chains of IgA, IgM and IgG of the human serum. With exceptions of the heavy chains of the immunoglobulins and α1-B-glycoprotein, all the other lectin-M-probed glycopeptides are acute-phase proteins. Subjecting human serum to immobilised-lectin-M affinity chromatography was able to isolate intact haptoglobin, α1-antitrypsin, α1B-glycoprotein, hemopexin and IgA. The use of champedak lectin-M to probe for serum glycoproteins that were separated in a 2-D gel electrophoresis and Western blotting technique may be conveniently applied to analyse.the acute-phase protein response. We have subsequently tested the methodology using the lectin to assess the human acute-phase response in patients with typhoid fever and leptospirosis. The study had focused on the simultaneous expression of four positive APP, i.e., haptoglobin (HP), hemopexin (HX), α1-antitrypsin (AT) and orosomucoid (OR), and two negative APP, i.e., transferrin (TF) and α2-HS glycoprotein (HS) in sera of ten typhoid and ten leptospirosis patients as opposed to ten normal healthy adult volunteers.  In the study involving sera from patients with typhoid fever, our data demonstrated the significant elevation of the percentage of volume contribution of all the four positive APP (p<0.01). However, the significant reduction of the percentage of volume contribution was only observed for TF but not HS. When volume ratios of the positive:negative APP were determined, significant enhancement of volume ratios of HP:TF, HX:TF, AT:TF and OR:TF in the sera of typhoid-afflicted patients was obtained. However, the volume ratios of all positive APP that were studied against HS were not significantly different between the two populations of normal subjects and typhoid patients. In the case of the leptospirosis patients, significant elevation (p<0.01) of the percentage of volume contribution was observed in both HP, a positive APP and  surprisingly, HS, a negative APP. As expected, significant reduction of the percentage of volume contribution was observed for TF. Similar to the typhoid-afflicted patients, significant enhancement of HP:TF, HX,TF, OR:TF and AT:TF was obtained and no significant difference between the normal and leptospirosia patients, of HP:HS, HX:HS, OR:HS and AT:HS volume ratios was observed.