Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type.

Abstract

The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with [3H]diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their aminoterminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.The discovery of an extralysosomal tripeptide-releasing amino-peptidase in rat liver has been reported (1). Its substrate specificity, as studied by use of a series of peptide substrates, is complex (1, 2). The peptidase releases tripeptides with little apparent similarity, although the rate of cleavage varies considerably. The enzyme, currently named tripeptidyl peptidase II (TPP-II) (2, 3), has a high native molecular weight (>106) (1), a subunit molecular weight of 135,000, and a neutral pH optimum (2). The presence ofTPP-II in numerous tissues of the rat (2) and in erythrocytes and liver of several other species (4) has been demonstrated. The human erythrocyte enzyme has been classified as a serine peptidase due to its complete inhibition by diisopropyl fluorophosphate (iPr2P-F) and phenylmethylsulfonyl fluoride (2). However, the enzyme was also inhibited by some of the thiol reagents tested, e.g., N-ethylmaleimide, p-chloromercuribenzoate, and Hg2+. TPP-II could be protected against iPr2P-F, phenylmethylsulfonyl fluoride, or N-ethylmaleimide by the use of a competitive inhibitor (2), suggesting that not only a serine, but also a cysteine residue is of importance for the activity of the enzyme. To obtain an unambiguous classification of TPP-II as a serine peptidase, we considered it necessary to isolate and determine the amino acid sequence of the part of the protein that contains the active serine residue. Information on the primary structure of the active site would also be ofinterest for the investigation of structural relationships between TPP-II and other peptidases. EXPERIMENTAL PROCEDURES Materials. [1,3-3H]iPr2P-F was purchased from AmershamInternational. Emulsifier scintillator 299 came from Packard. Dithiothreitol and bovine trypsin were from Boehringer Mannheim, and L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin...

Biosynthesis, Glycosylation, and Enzymatic Processingin Vivo of Human Tripeptidyl-peptidase I

“…Secretion of hTPP I expressed in the CHO cells we observed was also reported by others (22), although under standard conditions, endogenous hTPP I was not released by cultured primary cells (18). 2 Thus, selective secretion of hTPP I appears to result from its overexpression. The cellular mechanisms responsible for secretion of overexpressed hydrolases are still not understood.…”

“…1). TPP II is a cytosolic enzyme that belongs to the subtilisin subclass of serine peptidases (2). TPP I (EC 3.4.14.9) is a lysosomal exopeptidase with an acidic pH optimum (3,4) and a minor endoprotease activity (5).…”

Characterization and Cloning of Tripeptidyl Peptidase II from the Fruit Fly, Drosophila melanogaster

“…A Nucleosil C 18 column (10 m; 4 ϫ 250 mm) for HPLC was packed by Skandinaviska GeneTec AB (Kungsbacka, Sweden). Human TPP II was purified from red blood cells as described previously (8) with modifications subsequently reported (9,22). The inhibitor butabindide was a generous gift from Drs.…”

“…TPP II is a serine protease with a subtilisin-like catalytic domain, but compared with other subtilases, it contains an extended Cterminal region (9). The native form of the enzyme has a remarkably high molecular mass (Ͼ1000 kDa) that suggests an oligomeric association of the ϳ138-kDa subunits (7,8).…”

Mannose 6-Phosphorylated Proteins Are Required for Tumor Necrosis Factor-induced Apoptosis

“…Naturally occurring mutations in TPP1 are associated with a fatal lysosomal disorder, the classic late infantile form of neuronal ceroid lipofuscinosis (CLN2 or JanskyBielschowsky disease) (71,72), which is accompanied by a disturbed apoptosis (73). Of note, another tripeptidyl peptidase, TPP2, a cytoplasmic enzyme that belongs to the subtilisin class of serine peptidases (74), has been implicated in apoptosis induced by the bacterium Shigella flexneri and staurosporine in macrophages (75). Whether TPP1 is released in the cytosol and how this protease is connected to the cell death machinery needs to be clarified.…”