Guinea pig phospholipase B, identification of the catalytic serine and the proregion involved in its processing and enzymatic activity.
Guinea pig phospholipase B (GPPLB) is a glycosylated ectoenzyme of intestinal brush border membrane. It displays a broad substrate specificity and is activated by trypsin cleavage. The primary sequence contains four tandem repeat domains (I to IV) and several serines in lipase consensus sequences. We used site-directed mutagenesis to demonstrate that only the serine 399 present in repeat II is responsible for the various enzymatic activities of GPPLB. Furthermore, we characterized for the first time the retinyl esterase activity of the enzyme. We also constructed and expressed in COS-7 cells, an NH(2)-terminal repeat I deletion mutant which was detected at a very low level by immunoblot. However, confocal microscopy study showed a strong intracellular accumulation with a weak membrane expression of the mutated protein, indicating a role of the NH(2)-terminal repeat I in the processing of GPPLB. Nevertheless, the Western blot-detected protein presented a glycosylation and trypsin sensitivity patterns similar to wild type PLB. The mutant is also fully active without trypsin treatment, in contrast to native enzyme. Thus, we propose a structural model for GPPLB, in which the repeat I constitutes a lid covering the active site and impairing enzymatic activity, its removal by trypsin leading to an active protein.
Institut Fédératif de Recherche en Immunologie Cellulaire et Moléculaire, INSERM Unité 563, Centre de Physiopathologie de Toulouse Purpan, Département Lipoprotéines et Médiateurs Lipidiques, Hôpital Purpan, 31059 Toulouse Cedex, France.
Michel
M
Nauze
Lauriane
L
Gonin
Brigitte
B
Chaminade
Christine
C
Perès
Francoise
F
Hullin-Matsuda
Bertrand
B
Perret
Hugues
H
Chap
Ama
A
Gassama-Diagne
D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D020134:Catalytic Domain; D002462:Cell Membrane / Q000378:metabolism; D017353:Gene Deletion; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006168:Guinea Pigs; D015151:Immunoblotting; D007700:Kinetics; D008245:Lysophospholipase / Q000737:chemistry* / Q000378:metabolism*; D018613:Microscopy, Confocal; D008871:Microvilli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012694:Serine; D008245:Lysophospholipase; D006026:Glycoside Hydrolases; D014357:Trypsin