Guinea pig intestinal phospholipase B: protein expression during enterocyte maturation and effects of N-oligosaccharide removal on enzymatic activities and protein stability.
Guinea pig phospholipase B (PLB) is an intestinal brush-border hydrolase displaying a broad substrate specificity towards various dietary lipids. PLB was detected by immunoblotting as a single 140-kDa polypeptide in all cell populations isolated from guinea pig intestinal mucosa, but increased in parallel to its activity from undifferentiated to mature cells, the specific activity of the enzyme remaining constant. Moreover, N-glycosylation, which contributed to 23% of the apparent molecular mass, was identical along the cell differentiation axis. In all cell fractions, N-linked sugar chains were of the complex type, since they were removed by N-glycosidase F, whereas PLB remained insensitive to endoglycosidase H. Moreover, lack of O-glycosylation was demonstrated by the insensitivity of PLB to O-glycosidase and by its failure to interact with Helix pomatia lectin after prior treatment with neuraminidase or alpha-fucosidase. Enzymatic removal of sugar chains reduced phospholipase A2, lysophospholipase and diacylglycerol lipase activities by 27-35%, kinetic analysis indicating a decrease in apparent Vmax values for the three enzymatic activities, whereas the Km remained unchanged. Finally, the carbohydrate-depleted form of PLB did not display gross changes in thermal stability, in contrast to PLB from microorganisms previously investigated. Our data indicate that the high level of PLB N-glycosylation is poorly related to its biological function. Whether carbohydrate chains are involved in proper targeting of the enzyme to the brush-border membrane remains to be established.
Delagebeaudeuf
C
C
Institut Fédératif de Recherches en Immunologie Cellulaire et Moléculaire, Université Paul Sabatier, Toulouse, France.
Gassama
A
A
Collet
X
X
Nauze
M
M
Chap
H
H
D000818:Animals; D002454:Cell Differentiation; D004790:Enzyme Induction; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D006168:Guinea Pigs / Q000378:metabolism*; D007421:Intestine, Small / Q000201:enzymology* / Q000648:ultrastructure; D007700:Kinetics; D037102:Lectins / Q000378:metabolism; D008245:Lysophospholipase / Q000096:biosynthesis* / Q000737:chemistry / Q000187:drug effects / Q000235:genetics; D008871:Microvilli / Q000201:enzymology; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry*; D011489:Protein Denaturation
C038095:Helix lectin; D037102:Lectins; D009844:Oligosaccharides; D008245:Lysophospholipase; D006026:Glycoside Hydrolases