Is the bovine lysosomal phospholipase B-like protein an amidase?
The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B-like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 Å resolution. We showed that bPLBD1 has uniform noncomplex-type N-glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose-6-phosphorylation by a N-acetylglucosamine-1-phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N-glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N-acetylglucosamine-1-phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N-acetylglucosamine-1-phosphotransferase recognition. bPLBD1 is an N-terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn-hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn-hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family.
Institute of Biotechnology, University of Helsinki, FI-00014, Helsinki, Finland; Department of Biosciences, University of Helsinki, FI-00014, Helsinki, Finland.
Heidi
H
Repo
Elina
E
Kuokkanen
Esko
E
Oksanen
Adrian
A
Goldman
Pirkko
P
Heikinheimo
D000581:Amidohydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D002417:Cattle; D018360:Crystallography, X-Ray; D006367:HeLa Cells; D006801:Humans; D006860:Hydrogen Bonding; D007668:Kidney / Q000201:enzymology; D008245:Lysophospholipase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D008247:Lysosomes / Q000201:enzymology*; D008958:Models, Molecular; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein
D008245:Lysophospholipase; D000581:Amidohydrolases; C019665:amidase
Ntn-hydrolase; X-ray structure; glycosylation; lysosomal storage disorders; phosphotransferase