Affinity Purification and Characterization of a mannose specific Lectin and

α-mannosidase from the seeds of Annona squamosa

Supervisor: Prof. N. Siva Kumar

Department of Biochemistry,
University of Hyderabad.

Lectins, initially described as hemagglutinins, are now known as the glycoproteins of non-immune
origin with at least one sugar binding site per subunit. Lectins mediate many biological processes,
including cellular recognition, cell death, immune-modulation, cancer metastasis, cell adhesion, cell
migration. There is a growing evidence to support the role of plant lectins.
Also, Plant glycosidases are known to selectively edit glycosidic bonds, a property which serves to be
useful in therapeutics and drug designing.
A tropical plant Annona squamosa, is known for its medicinal effects, including insecticidal and
parasiticidal properties. The isolation, purification and biochemical characterization of biologically
important proteins present in the seeds of Annona squamosa were studied with a long-term objective of
understanding the structure-function relationships, and its biological significance.
A Lectin with a high affinity for glucose/mannose was isolated from Annona squamosa seeds
(Annonaceae) by affinity chromatography on seralose 4B Mannose affinity matrix, followed by gel
filtration on Sephacryl S-200; a two-step purification process.
SDS-PAGE analysis of purified Lectin under reducing conditions yielded three protein bands between
16 kDa to 33 kDa. However, only one band of Mr 54kDa was seen in non-reducing SDS PAGE. The
Lectin was a glycoprotein with 4.4% carbohydrate content(neutral sugar) and required divalent metal
cations (Ca2+, Mg2+, and Mn2+) for full activity. The Lectin weakly agglutinated human erythrocytes.
Annona Squamosa seeds contain several biologically important proteins among which α-mannosidase
was purified. α-Mannosidase is a key enzyme in the processing and degradation of N-glycans in plants
and animals.
α-Mannosidase from crude extracts of Annona squamosa (Indian Custard Apple) has been purified by
ammonium sulfate precipitation, phenyl Sepharose hydrophobic interaction chromatography, DE-52
anion exchange, gel filtration and Con A Sepharose chromatography.
The purified protein migrated as a single band corresponding to 230 kDa on NATIVE-PAGE analysis.
The pH and temperature optima of α-mannosidase activity was determined by use of p-nitrophenyl-α-
D-mannopyranoside as substrate. The purified enzyme is currently under further investigation for its
biochemical and biophysical properties.