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

There are 35 missense mutations among 68 different mutations in the TPP1 gene, which encodes tripeptidyl peptidase I (TPPI), a lysosomal aminopeptidase associated with classic late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). To elucidate the molecular mechanisms underlying TPPI deficiency in patients carrying missense mutations and to test the amenability of mutant proteins to chemical chaperones and permissive temperature treatment, we introduced individually 14 disease-associated missense mutations into human TPP1 cDNA and analyzed the cell biology of these TPPI variants expressed in Chinese hamster ovary cells. Most TPPI variants displayed obstructed transport to the lysosomes, prolonged half-life of the proenzyme, and residual or no enzymatic activity, indicating folding abnormalities. Protein misfolding was produced by mutations located in both the prosegment (p.Gly77Arg) and throughout the length of the mature enzyme. However, the routes of removal of misfolded proteins by the cells varied, ranging from their efficient degradation by the ubiquitin/proteasome system to abundant secretion. Two TPPI variants demonstrated enhanced processing in response to folding improvement treatment, and the activity of one of them, p.Arg447His, showed a fivefold increase under permissive temperature conditions, which suggests that folding improvement strategies may ameliorate the function of some misfolding TPPI mutant proteins.

Carbonic anhydrase II in the developing and adult human brain.

Carbonic anhydrase II (CA II) is one of 14 isozymes of carbonic anhydrases, zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. Mutations in CA II in humans lead to osteopetrosis with renal tubular acidosis and cerebral calcifications, a disorder often associated with mental retardation. Recently, new avenues in CA II research have opened as a result of discoveries that the enzyme increases bicarbonate and proton fluxes and may play an important role in brain tissue. In the human brain, CA II was localized to oligodendrocytes, myelin, and choroid plexus epithelium. Because this conclusion was based on a few fragmentary reports, we analyzed in more detail the expression of the enzyme in human telencephalon. By immunoblotting, we found a gradual increase in CA II levels from 17 weeks' gestation to childhood and adolescence. By immunohistochemistry, CA II was found to be present not only in oligodendrocytes and choroid plexus epithelium (declining with aging in both these locations), but also in a subset of neurons mostly with GABAergic phenotype, in a few astrocytes, and transiently during brain development in the endothelial cells of microvessels. The enzyme also occurred in oligodendrocyte processes in contact with myelinating axons, myelin sheaths, and axolemma, but was either absent or appeared in minute amounts in compact myelin. These findings suggest the possible involvement of CA II in a wide spectrum of biologic processes in the developing and adult human brain and may contribute to better understanding of the pathogenesis of cerebral calcifications and mental retardation caused by CA II deficiency.

Ser475, Glu272, Asp276, Asp327, and Asp360 are involved in catalytic activity of human tripeptidyl-peptidase I.

Tripeptidyl-peptidase I (TPP I) is a lysosomal aminopeptidase that sequentially removes tripeptides from small polypeptides and also shows a minor endoprotease activity. Mutations in TPP I are associated with a fatal lysosomal storage disorder--the classic late-infantile form of neuronal ceroid lipofuscinoses. In the present study, we analyzed the catalytic mechanism of the human enzyme by using a site-directed mutagenesis. We demonstrate that apart from previously identified Ser475 and Asp360, also Glu272, Asp276, and Asp327 are important for catalytic activity of the enzyme. Involvement of serine, glutamic acid, and aspartic acid in the catalytic reaction validates the idea, formulated on the basis of significant amino acid sequence homology and inhibition studies, that TPP I is the first mammalian representative of a growing family of serine-carboxyl peptidases.