Abstract
Tripeptidyl-peptidase I (TPP I) is a lysosomal serine-carboxyl peptidase that sequentially removes tripeptides from polypeptides. Naturally occurring mutations in TPP I are associated with the classic late infantile neuronal ceroid lipofuscinosis. Human TPP I has five potential N-glycosylation sites at Asn residues 210, 222, 286, 313, and 443. To analyze the role of N-glycosylation in the function of the enzyme, we obliterated each N- glycosylation consensus sequence by substituting Gln for Asn, either individually or in combinations, and expressed mutated cDNAs in Chinese hamster ovary and human embryonic kidney 293 cells. Here, we demonstrate that human TPP I in vivo utilizes all five N-glycosylation sites. Elimination of one of these sites, at Asn-286, dramatically affected the folding of the enzyme. However, in contrast to other misfolded proteins that are retained in the endoplasmic reticulum, only a fraction of misfolded TPP I mutant expressed in Chinese hamster ovary cells, but not in human embryonic kidney 293 cells, was arrested in the ER, whereas its major portion was secreted. Secreted proenzyme formed non-native, interchain disulfide bridges and displayed only residual TPP I activity upon acidification. A small portion of TPP I missing Asn-286-linked glycan reached the lysosome and was processed to an active species; however, it showed low thermal and pH stability. N-Glycans at Asn-210, Asn-222, Asn-313, and Asn-443 contributed slightly to the specific activity of the enzyme and its resistance to alkaline pH-induced inactivation. Phospholabeling experiments revealed that N-glycans at Asn-210 and Asn-286 of TPP I preferentially accept a phosphomannose marker. Thus, a dual role of oligosaccharide at Asn-286 in folding and lysosomal targeting could contribute to the unusual, but cell type-dependent, fate of misfolded TPP I conformer and represent the molecular basis of the disease process in subjects with naturally occurring missense mutation at Asn-286.
Highlights
Tripeptidyl-peptidase I (TPP I)1 (EC 3.4.14.9) is a lysosomal aminopeptidase that sequentially removes tripeptides from polypeptides with an unsubstituted N terminus and uncharged
Human TPP I Contains Five N-Linked Glycans, of Which One at Asn-286 Is Involved in Maturation of the Enzyme— Human TPP I has five potential N-glycosylation sites at Asn residues 210, 222, 286, 313, and 443; they are all located in the mature enzyme
To determine which of these sites are utilized in vivo, we eliminated each potential N-glycosylation site in TPP I cDNA by converting the Asn codon to a Gln codon
Summary
Tripeptidyl-peptidase I (TPP I)1 (EC 3.4.14.9) is a lysosomal aminopeptidase that sequentially removes tripeptides from polypeptides with an unsubstituted N terminus and uncharged. Our study shows that all five potential Nglycosylation consensus sites are utilized by human TPP I in vivo and that Asn-286-linked glycan is essential for both folding and lysosomal targeting of the enzyme.
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