| Laine Lab: Congratulations on
travel awards. Markus Hardt and Marianne McKee (Ph.D.,
August 2001 in analytical chemistry) of Roger Laine's group have won $750
Travel Awards from the Society for Glycobiology, presentation of papers
to be made at the November, 2001 Meeting in San Francisco. Abstacts
of the papers follow:
M. Hardt and R.A.
Laine. Site directed mutagenesis of the chitin-binding domain from
B.
circulans chitinase A1 and analysis by a green fluorescent protein-based
binding assay.
Abstract: A fluorescent
binding assay was developed to investigate the effects of site directed
mutagenesis on the binding affinity and binding specificity
of the chitin-binding domain of chitinase A1 from Bacillus circulans
WL-12. The chitin-binding domain was genetically fused to the N-terminus
of the green fluorescent protein, GFP. The polyhistidine tagged hybrid
protein was expressed in Escherichia coli under the dose-dependent
regulation of the araBAD promoter and purified using metal affinity chromatography.
Residues that have been suggested to be involved in binding from
previous three-dimensional studies were site-specifically mutated. Purified
fusion proteins were incubated with various insoluble polysaccharides.
The polysaccharides and the bound protein were removed by centrifugation.
The free protein concentration was measured fluorometrically. Scatchard
plots of the adsorption data were used for analysis. The fusion protein
proved to be useful for specifically labeling cell walls of fungi
and yeast.
M. McKee, B.C.R. Zhu, and
R.A.
Laine. Improved Assay for GDP-Mannose 4,6-dehydratase.
Abstract: GDP-Mannose 4,6-dehydratase
(4,6D) is the first of a 2-enzyme process in the biosynthesis of
GDP-L-fucose. The second enzyme is a "reductase-epimerase." The intermediate
after the 4,6-dehydratase reaction is GDP-4-keto, 6-deoxy-D-mannose. Fucose
is a crucial part of the epitope for Sialyl-LeX, the selectin address for
neutrophil extravasation. LAD-2 human individuals of the Bombay blood type
who apparently produce no fucose are abnormal, retarded, cretinized and
of short stature, having frequent respiratory infections and a partial
inability to fend of infections. Therefore this enzyme system is important
for normal development and immune function. In the interest of studying
the activity of 4,6D, we undertook to develop a non-radiolabel assay. HPLC
of sugar nucleotides and derivatives is common, however, separation of
GDP-D-Mannose, GDP, GMP and GDP-L-fucose had not been accomplished. Using
Nucleosil columns after attempts with others, we discovered that a simple
pH adjustment allowed complete separation of all of these compounds. In
addition, GDP-D-Mannose and GDP-4-keto-6-deoxy-D-Mannose were completely
separated, allowing a clean assay for 4,6D. The product was characterized
by reduction, acid cleavage and acetylation to be D-Rhamnose/D-Talose,
confirming its identity. Fucose and 2-deoxyfuconojirimycin were found to
be mild inhibitors with Ki values near 400µM, while GDP-L-fucose
inhibited the enzyme at a Ki of 90µM. |
