The effect of the carbohydrate binding module on substrate degradation by the human chitotriosidase

Two DNA plasmids were transfected into HEK-293-6E grown in F17 medium supplemented with Kolliphor P188 (0.1%). Transfection was performed with PEIpro when the cell density in the cultures was 1.7 x 10^6 cells/mL. 48 h after transfection, tryptone N1 feeding medium was added to a final concentration of 0.5% and harvesting of the protein was performed 96h after transfection.

Human chitotriosidase (HCHT) is one of two active glycoside hydrolase family 18 chitinases produced by humans. The enzyme is associated with several diseases and is thought to play a role in the anti-parasite responses of the innate immune system. HCHT occurs in two isoforms, one 50 kDa (HCHT50) and one 39 kDa variant (HCHT39). Common for both isoforms is a catalytic domain with the (beta/alpha)8 TIM barrel fold. HCHT50 has an additional linker-region, followed by a C-terminal carbohydrate-binding module (CBM) classified as CBM family 14 in the CAZy database. To gain further insight into enzyme functionality and especially the effect of the CBM, we expressed both isoforms and compared their catalytic properties on chitin and high molecular weight chitosans. HCHT50 degrades chitin faster than HCHT39 and much more efficiently. Interestingly, both HCHT50 and HCHT39 show biphasic kinetics on chitosan degradation where HCHT50 is faster initially and HCHT39 is faster in the second phase. Moreover, HCHT50 produces distinctly different oligomer distributions than HCHT39. This is likely due to increased transglycosylation activity for HCHT50 due the CBM extending the positive subsites binding surface and therefore promoting transglycosylation. Finally, studies with both chitin and chitosan showed that both isoforms have a similarly low degree of processivity. Combining functional and structural features of the two isoforms, it seems that HCHT combines features of exo-processive and endo-nonprocessive chitinases with the somewhat unusual CBM14 to reach a high degree of efficiency, in line with its alleged physiological task of being a "complete" chitinolytic machinery by itself.