Variation of the glycosylation of human pancreatic bile-salt-dependent lipase.

International audienceGlycoproteins of human pancreatic juice were characterized by means of lectins after electrophoresis and electrotransfer to nitrocellulose membranes. For the detected glycoproteins, only a 100-kDa glycoprotein varied in the pancreatic juice from a normal patient (i.e. without any pancreatic disorder) compared to the pancreatic juice from a patient suffering from chronic pancreatitis. This protein, which is the only protein in human pancreatic juice which is O-glycosylated and N-glycosylated, was identified as the bile-salt-dependent lipase. Among the glycosylated proteins present in human pancreatic juice, only the glycosylation of bile-salt-dependent lipase differs between individuals. The enzyme was isolated either from normal or pathological human pancreatic juices. The purified variants have an identical molecular mass and amino-acid composition. As suspected from lectin affinity studies, the oligosaccharide composition differs between the variants. The structure of the N-linked oligosaccharides of the variant from the pancreatic juice of a normal donor correlated with complete processing and maturation of a complex-type N-glycan. Alteration of the maturation process can be detected for a bile-salt-dependent-lipase variant from a patient suffering with chronic pancreatitis, since the carbohydrate composition is compatible with the predominance of hybrid or high-mannose-type structures. The amount of sugar involved in O-glycosylation associated with the peanut agglutinin reactivity suggests the presence of 12-14 minimal Gal beta 1-->3GalNac-->T/S O-glycan structures which are sialylated and fucosylated. The amount of sugar involved in the O-linked oligosaccharide structure appears to be unchanged in the variants isolated from the pathological pancreatic juice

Investigation of two glycosylated forms of bile-salt-dependent lipase in human pancreatic juice.

International audiencePure human pancreatic bile-salt-dependent lipase, devoid of its oncofetal glycoform [Mas, E., Abouakil, N., Roudani, S., Miralles, F., Guy-Crotte., O., Figarella, C., Escribano, M. J. & Lombardo, D. (1993) Biochem. J. 289, 609-615], was analyzed on immobilized concanavalin A (ConA). Two variants were separated: an unabsorbed ConA-unreactive fraction; and an absorbed ConA-reactive fraction. Carbohydrate compositions of ConA-reactive and ConA-unreactive fractions were not significantly different, and analysis of 3H-labelled oligosaccharides liberated from these fractions on the ConA-Sepharose column indicated that the fractionation of the bile-salt-dependent lipase on this column depends upon oligosaccharide structures. The activity of the ConA-reactive fraction was however much lower, independent of the substrate (4-nitrophenyl hexanoate or cholesteryl esters), than that of the ConA-unreactive fraction. Therefore, catalytic constants for the hydrolysis of 4-nitrophenyl hexanoate were determined; both fractions had quite similar Km, while the kcat for the ConA-unreactive fraction was 3-4-fold higher than that of the ConA-reactive fraction. ConA-reactive and ConA-unreactive fractions were shown to have slightly different molecular masses and different amino acid compositions. Cleavage patterns after cyanogen bromide treatment of the ConA-reactive and ConA-unreactive fractions suggested that the ConA-reactive (high Mr form) and ConA-unreactive (low Mr form) forms could be different isoforms of the bile-salt-dependent lipase secreted by the human pancreas