RECOMBINANT HUMAN-MILK BILE-SALT-STIMULATED LIPASE - FUNCTIONAL-PROPERTIES ARE RETAINED IN THE ABSENCE OF GLYCOSYLATION AND THE UNIQUE PROLINE-RICH REPEATS

Human milk bile-salt-stimulated lipase ensures efficient utilization o f milk lipid in breast-fed infants. The N-terminal two-thirds of the p eptide chain is highly conserved and shows striking similarities to ty pical esterases. In contrast, the remaining C-terminal part consists o f a unique sequence of 16 proline-rich O-glycosylated repeats of 11 re sidues each. Recently we could show, using recombinant Lipase variants , that neither these repeats nor the single N-linked sugar chain are e ssential for catalytic efficiency. In the present study, we report on the lack of importance of glycosylation and the unique repeats for oth er important functional properties, i.e. bile-salt activation, heparin binding, heat stability, stability at low pH and resistance to proteo lytic inactivation. Compared to native enzyme, recombinant full-length lipase produced in two mammalian cell lines differed slightly in glyc osylation pattern with no effects on the functional properties. Moreov er, a variant lacking all repeats and the C-terminal tail following th e last repeat exhibited the same functional characteristics as purifie d native milk enzyme. Thus, the structural basis for all the typical a nd functionally important properties reside in the N-terminal conserve d part, in spite of the fact that none of these properties are shared by typical esterases. We could however, demonstrate that the C-termina l repeats are responsible for the unusual behaviour of the enzyme in s ize-exclusion chromatography, resulting in a considerably higher than expected apparent molecular mass.