Structure of Tripeptidyl-peptidase I Provides Insight into the Molecular Basis of Late Infantile Neuronal Ceroid Lipofuscinosis

Abstract

Late infantile neuronal ceroid lipofuscinosis, a fatal neurodegenerative disease of childhood, is caused by mutations in the TPP1 gene that encodes tripeptidyl-peptidase I. We show that purified TPP1 requires at least partial glycosylation for in vitro autoprocessing and proteolytic activity. We crystallized the fully glycosylated TPP1 precursor under conditions that implied partial autocatalytic cleavage between the prosegment and the catalytic domain. X-ray crystallographic analysis at 2.35 Å resolution reveals a globular structure with a subtilisin-like fold, a Ser 475 -Glu 272 -Asp 360 catalytic triad, and an octahedrally coordinated Ca 2؉ -binding site that are characteristic features of the S53 sedolisin family of peptidases. In contrast to other S53 peptidases, the TPP1 structure revealed steric constraints on the P4 substrate pocket explaining its preferential cleavage of tripeptides from the unsubstituted N terminus of proteins. Two alternative conformations of the catalytic Asp 276 are associated with the activation status of TPP1. 28 disease-causing missense mutations are analyzed in the light of the TPP1 structure providing insight into the molecular basis of late infantile neuronal ceroid lipofuscinosis.

Functional consequences and rescue potential of pathogenic missense mutations in tripeptidyl peptidase I

“…All three of these residues also provide H-bonds to the adjacent part of the prosegment; thus, those contacts would be affected as well. Cys365 participates in an intramolecular disulfide bond formation with Cys526 [Guhaniyogi et al, 2009;Pal et al, 2009]. Therefore, the assisted folding process of this variant might be prolonged because of the inability of the ER machinery to form a native disulfide bond.…”

“…The effects of mutations on the tertiary structure of TPPI were analyzed on a visual level with the Swiss-PdbViewer [Guex and Peitsch, 1997] using protein structure files PDB 3EDY [Guhaniyogi et al, 2009] and PDB 3EE6 [Pal et al, 2009].…”

“…Mutagenic analyses and inhibition studies identified residues Ser475, Glu272, Asp276, Asp360, and Asp327 as being closely involved in catalytic activity and substrate binding, with Ser475 acting as an active-site nucleophile Oyama et al, 2005;Walus et al, 2005]. Recently, two independent teams of researchers published crystal structures of TPPI proenzyme, which helped refine the mechanisms of activation and tripeptidase activity of the enzyme [Guhaniyogi et al, 2009;Pal et al, 2009].…”

Crystal Structure and Autoactivation Pathway of the Precursor Form of Human Tripeptidyl-peptidase 1, the Enzyme Deficient in Late Infantile Ceroid Lipofuscinosis

“…Biomedical Relevance-The structure of pro-TPP1 provides some insights into disease-causing mutations in LINCL (discussed in the accompanying paper by Pal et al (81) (supplemental Fig. S8).…”

Impaired Lysosomal Trimming of N-Linked Oligosaccharides Leads to Hyperglycosylation of Native Lysosomal Proteins in Mice with α-Mannosidosis

“…On the other hand, it was demonstrated for recombinant human NPC2 protein that the diglycosylated NPC2 protein exhibited significantly lower cholesterol transfer rates (three-to fourfold) than the monoglycosylated NPC2 variant, most likely due to charge repulsions between the oligosaccharides and anionic groups of the phospholipids (27). Comparably, it was shown that the deglycosylation of the lysosomal ␤-glucosidase as well as TPP1 leads to a complete loss of enzyme activity (20,35). Hyperglycosylation of N-linked oligosaccharides due to an impaired trimming had been described for Golgi ␣-mannosidase II deficiency, also called HEMPAS (hereditary erythroblastic multinuclearity with a positive acidified serum lysis test), leading to a reduced ability to form complex-type oligosaccharides (11).…”