Is the bovine lysosomal phospholipase B-like protein an amidase?

• Published in: Proteins, structure, function, and bioinformatics structure, function, and bioinformatics, 2014-02, Vol.82 (2), p.300-311
• Main Authors: Repo, Heidi, Kuokkanen, Elina, Oksanen, Esko, Goldman, Adrian, Heikinheimo, Pirkko
• Format: Article
• Language: English
• Subjects: Amidase; Amidohydrolases - chemistry; Amidohydrolases - isolation & purification; Amidohydrolases - metabolism; Animals; Catalytic Domain; Cattle; Crystallography, X-Ray; glycosylation; HeLa Cells; Humans; Hydrogen Bonding; Kidney - enzymology; Lysophospholipase - chemistry; Lysophospholipase - isolation & purification; Lysophospholipase - metabolism; lysosomal storage disorders; Lysosomes - enzymology; Models, Molecular; Ntn-hydrolase; phosphotransferase; Protein Processing, Post-Translational; Protein Structure, Quaternary; Protein Structure, Secondary; Sequence Analysis, Protein; X-ray structure
• ISSN: 0887-3585; 1097-0134
• DOI: 10.1002/prot.24388

ABSTRACT 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. Proteins 2014; 82:300–311. © 2013 Wiley Periodicals, Inc.