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12225.Full.Pdf 2626 Corrections Proc. Natl. Acad. Sci. USA 93 (1996) Developmental Biology. In the article ‘‘In vitro generation of Cell Biology. In the article ‘‘Purification, cDNA cloning, hematopoietic stem cells from an embryonic stem cell line’’ by functional expression, and characterization of a 26-kDa en- Ronald Palacios, Eva Golunski, and Jacqueline Samaridis, dogenous mammalian carboxypeptidase inhibitor’’ by Emman- which appeared in no. 16, August 1, 1995, of Proc. Natl. Acad. uel Normant, Marie-Pascale Martres, Jean-Charles Schwartz, Sci. USA (92, 7530–7534), the histograms in Fig. 2 B and C (on and Claude Gros, which appeared in number 26, December 19, p. 7532) were incorrect. The corrected Fig. 2 is illustrated 1995, of Proc. Natl. Acad. Sci. USA (92, 12225–12229), the below. The authors apologize for any inconvenience this may authors request the following correction be noted. The Gen- have caused readers. Bank Accession No. on p. 12225 should read U40260. FIG. 2. Specificity of the 28-8-6 antibody for MHC class I H-2b haplotype. The MHC class I H-2b-specific antibody 28-8-6 stains mononuclear marrow cells from C57BLy6 (H-2b) (A) but not from CB17 SCID (H-2D) (B) or C3H SCID (H-2K) (C) mice. Downloaded by guest on September 23, 2021 Proc. Natl. Acad. Sci. USA Vol. 92, pp. 12225-12229, December 1995 Cell Biology Purification, cDNA cloning, functional expression, and characterization of a 26-kDa endogenous mammalian carboxypeptidase inhibitor EMMANUEL NORMANT, MARIE-PASCALE MARTRES, JEAN-CHARLES SCHWARTZ, AND CLAUDE GROS* Unite de Neurobiologie et Pharmacologie (U. 109) de l'Institut National de la Sante et de la Recherche Medicale, Centre Paul Broca, 2ter rue d'Alesia, 75014 Paris, France Communicated by William N. Lipscomb, Harvard University, Cambridge, MA, August 4, 1995 ABSTRACT The recent demonstration of the occurrence propeptide was characterized but no CPA activity could be in rat brain and other nonpancreatic tissues of carboxypep- generated after incubation of tissue extracts in presence of tidase A (CPA) gene transcripts without associated catalytic trypsin under conditions that should have severed the "acti- activity could be ascribed to the presence of a soluble endog- vation segment" and released the mature CPA. The discrep- enous protein inhibitor. This tissue carboxypeptidase inhib- ancy was explained, in this case, by detection, in extracts from itor (TCI), detected by the inhibition of added bovine pancre- brain and other tissues expressing the CPA1 gene, of an atic CPA, was purified from rat brain. Peptides were obtained endogenous inhibitory activity directed toward added bovine by partial proteolysis of purified TCI, a protein of "30 kDa, pancreatic CPA. The latter was rather thermostable but sen- and starting from their sequences, a full-length cDNA encod- sitive to the action of Pronase. ing a 223-amino acid protein containing three potential In the present work, we have purified this tissue carboxypep- phosphorylation sites was cloned from a cDNA library. Its tidase inhibitor (TCI) from rat brain, cloned the corresponding identity with TCI was shown by expression in Escherichia coli cDNA,t and studied its properties and tissue distribution. of a recombinant protein recognized by antibodies raised against native TCI and displaying characteristic CPA- inhibiting activity. TCI appears as a hardly reversible, non- MATERIALS AND METHODS competitive, and potent inhibitor of CPA1 and CPA2 (K; 3 Materials. CPA (from bovine pancreas, 50 units/mg of nM) and mast-cell CPA (K1 = 16 nM), which is less potent protein), CPB (from porcine pancreas, 105 units/mg of pro- against carboxypeptidase B (CPB; Ki = 194 nM) and inactive tein), carboxypeptidase Y (CPY, from baker's yeast, 100 on various other proteases. This pattern ofselectivity might be units/mg of protein), leucine aminopeptidase (from porcine attributable to a limited homology of a 11-amino acid se- pancreas, 140 units/mg of protein), phenylmethylsulfonyl flu- quence with sequences within the activation segments of CPA oride, o-phthaldialdehyde, hippuryl-L-phenylalanine (HF), N- and CPB known to interact with residues within their active ethylmaleimide, and leupeptin were supplied by Sigma; ami- sites. The widespread expression ofTCI in a number oftissues nopeptidase M (from hog kidney, 20 units/mg of protein) was (e.g., brain, lung, or digestive tract) and its apparently cyto- from Pierce; a-chymotrypsin (from porcine pancreas, 20 solic localization point to a rather general functional role, e.g., units/mg of protein), trypsin (from bovine pancreas, 10,900 in the control of cytosolic protein degradation. units/mg of protein), and elastase (from porcine pancreas, 85 units/mg of protein) were from Boehringer Mannheim; and Proteinases participate in a large variety of cell processes, Ala-AMC and other AMC substrates were from Bachem namely, in modeling and communication, but their activity (AMC is 7-amido-4-methylcoumarin). could be detrimental if not tightly controlled. The activity of Radioactive materials were from Amersham and other many proteinases is controlled by the occurrence of endoge- materials were from commercial sources. nous protein inhibitors as is the case for matrix metal- Enzyme Activity Determinations. CPA activity was deter- loproteinases with which such inhibitors may form hardly mined by using the radioiodinated Bolton-Hunter reagent reversible complexes (1). For other proteinases, the control derivative of the dipeptide Arg-Phe as substrate (8). Briefly, mechanism is the necessary cleavage of a segment of a samples (50 ,ul) were incubated with radioiodinated Bolton- propeptide to release the mature proteinase, endowed with the reac- catalytic activity, a scheme that applies, for instance, to the Hunter reagent-coupled Arg-Phe (105 dpm, 50 1,l), pancreatic carboxypeptidases (2-4). tion was stopped by adding 0.2 M HCl (50 ,ul), and liberated These two major control mechanisms are not mutually 125I-labeled Bolton-Hunter reagent was recovered by reverse- exclusive but, for carboxypeptidases A and B (CPA and CPB, phase chromatography (Porapak Q, Millipore). Alternatively, respectively), the only known protein inhibitors-i.e., the the substrate hippuryl-[3H]phenylalanine ([3H]HF, 105 inhibitors from Solanacea (5, 6) or Ascaris lumbricoides (7)- dpm/50 ,ul) was used, and [3H]phenylalanine formed in pres- are not of mammalian origin. ence of CPA was also isolated. CPA activity was also deter- We have recently characterized in brain and several other mined fluorimetrically, with HF as substrate and o-phthal- extrapancreatic tissues the presence of CPA1 and CPA2 gene dialdehyde as fluorogenic reagent (9). transcripts that were not accompanied, however, by any typical CPB (EC 3.4.17.2), carboxypeptidase H (CPH, EC 3.4.17.10), CPA catalytic activity, even using extremely sensitive detection carboxypeptidase M (CPM, EC 3.4.17.12), and CPY (EC systems (8). For CPA2, this discrepancy was explained by the 3.4.16.14) activities were measured with hippuryl-[3H]arginine as fact that the transcript corresponds to a splice variant leading to a shorter CPA2 isoform with modified CPA activity. In Abbreviations: CPA, CPB, etc., carboxypeptidase A, carboxypeptidase contrast, for CPA1, a mRNA encoding the full-size pancreatic B, etc.; TCI, tissue carboxypeptidase inhibitor; HF, hippuryl-L-phenyl- alanine; AMC, 7-amido-4-methylcoumarin; RT-PCR, reverse tran- scription-coupled PCR. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in tThe sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U10260). 12225 12226 Cell Biology: Normant et al. Proc. Natl. Acad. Sci. USA 92 (1995) substrate and [3H]arginine was isolated on a polystyrene bead blunt-ended with T4 DNA polymerase, phosphorylated, and column with water as eluent. CPH activity was measured from subcloned in a Sma I-digested pGEM-4Z. AtT20 cells culture medium (10). CPM activity was assayed from A probe generated by reverse transcription-coupled PCR human lung membranes (11). CPY activity was assayed in 50 mM (RT-PCR) from rat brain poly(A)+ mRNA by using degen- potassium phosphate (pH 6.0) using the commercial enzyme. erated primers designed from peptide 2 (see Fig. 3) was used Neprilysin (enkephalinase, EC 3.4.24.11) activity was eval- to screen a rat hypothalamic cDNA library (1 X 106 phages) uated as described (12). Peptidyl dipeptide hydrolase (angio- constructed on AZAP II (Stratagene). This probe (1 X 106 tensin-converting enzyme, EC 3.4.15.1) activity was evaluated dpm/ml) was hybridized in 40% (vol/vol) formamide/4X from human kidney membranes with 0.2 mM o-aminobenzoyl- SSC/lx Denhardt's solution/8 mM Tris-HCl, pH 7.4/yeast glycyl-p-nitrophenylalanyl proline as substrate (13). Amino- tRNA (20 ,ug/ml)/0.1% SDS/salmon sperm DNA (20 ,ug/ peptidase M (EC 3.4.11.2), leucine aminopeptidase (EC ml)/10% (wt/vol) dextran sulfate. Clones were plaque- 3.4.11.1), and leukotriene A4 hydrolase (EC 3.3.2.6) amino- purified and Bluescript KS(+) plasmids containing cDNA peptidase activities were assayed from commercial enzymes inserts were recovered using the helper phage R508 and with 25 ,uM Ala-AMC in 50 mM Tris HCl (pH 7.4). Trypsin sequenced (15). The sequence of 3'-end region was completed (EC 3.4.21.4), a-chymotrypsin (EC 3.4.21.1), and elastase (EC by using a rapid amplification of cDNA ends system (16). 3.4.21.36) activities were assayed with 2.5 ,AM Z-Arg-AMC, Expression in Escherichia coli. An 890-bp PCR fragment Ala-Ala-Phe-AMC, and Suc-Ala-Ala-Ala-AMC as substrates, containing the entire open reading frame was subcloned in respectively (where Z is benzyloxycarbonyl and Suc is succi- pUC9 plasmid.
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