| Summary: | Endoglucanase A (CenA), a component o f the multi-enzyme cellulase system o f the
bacterium Cellulomonas fimi (C. fimi), is typical of many cellulases, being a multi-domain
protein. CenA has two domains which have been well characterized, a catalytic domain
and a cellulose-binding domain (CBD) separated by a proline, threonine rich linker (PT
linker). This study investigated the structure, function and possible applications of this
interdomain linker.
Modification of the PT linker's size and composition gave insights into the effect of
the linker on hydrolysis of a variety of cellulosic substrates and the nature of its
susceptibility to proteolysis. Modifying the linker size and composition had little effect on
the hydrolysis rate. Partial deletion reduced activity on crystalline and amorphous
celluloses, whereas doubling or replacing it with the fibronectin type III (Fn3) repeats from
the endoglucanase B increased the rate of hydrolysis of crystalline cellulose. Altering the
linker did not affect activity on 2',4'-dinitrophenyl β-D-cellobioside (2,4-DNPC) except
for the Fn3 construct which showed decreased activity. A sequence susceptible to papain
and C. fimi protease was defined which could be modified to prevent proteolysis. This has
potential implications for the design of linkers for stable CBD-fusion proteins.
Examining the glycosylation of CenA produced by C. fimi and Streptomyces
lividans (S. lividans) gave insights into eubacterial protein glycosylation. Glycosylation
was confined to the PT linker at sites conforming to an O-glycosylation consensus
sequence for some mammalian glycoproteins. The oligosaccharide composition was
dependent on the host, but the glycosylation mechanism is likely similar as they
discriminated between the same protein features. Oligosaccharide size and linkage
information for biose units of S. lividans produced proteins were determined, the latter by a
novel application of fluorophore-assisted carbohydrate electrophoresis. Glycosylation
slowed proteolysis and slightly reduced the activity on 2,4-DNPC.
The CenA linker resembles the hinge of human immunoglobulin A1 (IgA1). A
hybrid protein, CenAIgA1h - where the PT linker was replaced by the IgA1 hinge- and
CenA were useful substrates for probing the specificity of a number of IgA1 proteases
produced by a variety of pathogenic bacteria. The most striking finding was that only one
of two IgA1 proteases which have high sequence identity and cleave the identical bond in
IgA1, cleaved CenA. These substrates will be useful in future studies of IgA1 protease
specificity.
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