From the Departamento de Bioquímica y Biología Molecular and § Biología Funcional, Facultad de Medicina, Universidad de Oviedo, 33006-Oviedo, Spain
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ABSTRACT |
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We have identified and characterized a novel human cysteine proteinase of the papain family. A full-length cDNA for this enzyme was cloned from a human brain cDNA library. Nucleotide sequence analysis revealed that the isolated cDNA codes for a polypeptide of 303 amino acids, tentatively called cathepsin Z, that exhibits structural features characteristic of cysteine proteinases. Fluorescent in situ hybridization experiments revealed that the human cathepsin Z gene maps to chromosome 20q13, a location that differs from all cysteine proteinase genes mapped to date. The cDNA encoding cathepsin Z was expressed in Escherichia coli as a fusion protein with glutathione S-transferase, and after purification, the recombinant protein was able to degrade the synthetic peptide benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin, used as a substrate for cysteine proteinases. Northern blot analysis demonstrated that cathepsin Z is widely expressed in human tissues, suggesting that this enzyme could be involved in the normal intracellular protein degradation taking place in all cell types. Cathepsin Z is also ubiquitously distributed in cancer cell lines and in primary tumors from different sources, suggesting that this enzyme may participate in tumor progression as reported for other cathepsins. Finally, on the basis of a series of distinctive structural features, including diverse peptide insertions and an unusual short propeptide, together with its unique chromosomal location among cysteine proteinases, we propose that cathepsin Z may be the first representative of a novel subfamily of this class of proteolytic enzymes.
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INTRODUCTION |
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The cysteine proteinases belonging to the papain family represent a major component of the lysosomal proteolytic system and play an essential role in protein degradation and turnover (1, 2). In addition, these proteolytic enzymes appear to play an extracellular role in a number of normal and pathological conditions including bone remodeling (3), prohormone activation (4), rheumatoid arthritis (5), Alzheimer's disease (6), pulmonary emphysema (7), and cancer invasion and metastasis (8). To date, nine human cysteine proteinases of the papain family have been isolated and characterized at the amino acid sequence level: cathepsin B (9), cathepsin L (10, 11), cathepsin H (12, 13), cathepsin S (14, 15), cathepsin C (16), cathepsin O (17), cathepsin K (18, 19), cathepsin W (20), and cathepsin L2 (21). All of them contain an essential cysteine residue in their active site but differ in tissue distribution and in some enzymatic properties, including substrate specificities and pH stability. Furthermore, several groups have reported the existence of additional cysteine proteinases including cathepsins M, N, P, and T, which were originally identified because of their degrading activity on specific substrates such as aldolase, collagen, proinsulin, or tyrosine aminotransferase, but whose characterization at the molecular level has not yet been reported (22-25).
According to structural and functional data, it is well established that the different cysteine proteinases of the papain family are synthesized as preproenzymes, which are processed to the corresponding proenzymes and targeted to the lysosomes by the mannose 6-phosphate signal attached to them. The enzymes are further processed to mature forms consisting of either a single polypeptide chain or a two-chain form composed of heavy and light chains linked by a disulfide bond (26). However, in some cases, the precursors of these lysosomal enzymes escape from this processing pathway and continue along the secretory route, entering storage granules and being finally released into the extracellular space (27). In fact, a series of reports have shown that several lysosomal cysteine proteinases are released by tumor cells from different sources, supporting the concept that secreted lysosomal enzymes may play important roles in the developmnent of malignant processes (28-30). Furthermore, lysosomal cysteine proteinases have been found to be secreted by activated macrophages, by osteoclasts, or by fibroblasts from patients with I-cell disease, thereby extending the pattern of physiological and pathological conditions in which these enzymes may be involved (2, 27). Based on the hypothesis that a number of different cysteine proteinases could be responsible for the wide variety of biological functions ascribed to this protein family, we have been interested in examining the possibility that additional yet uncharacterized family members could be produced by human tissues. This search for new human cysteine proteinases led us to identify cathepsin O, originally cloned from a breast carcinoma but widely distributed in human tissues (17). We have also described the cloning and characterization of human bleomycin hydrolase, a cytosolic cysteine proteinase distantly related to other members of the papain family and involved in chemotherapy resistance (31, 32). Finally, we have recently reported the finding of cathepsin L2, a cysteine proteinase structurally related to cathepsin L, but showing a unique tissue distribution (21). In this study, we report the identification, chromosomal location, and structural and enzymatic characterization of a novel member of this family of enzymes, which has been tentatively called cathepsin Z.
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EXPERIMENTAL PROCEDURES |
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Materials--
Restriction endonucleases and other reagents used
for molecular cloning were from Boehringer Mannheim (Mannheim,
Germany). Human brain and prostate cDNA libraries, constructed in
gt11, and Northern blots containing poly(A)+ RNAs
prepared from different human tissues and cancer cell lines were
purchased from CLONTECH (Palo Alto, CA).
Double-stranded DNA probes were radiolabeled with
[
-32P]dCTP (3000 Ci/mmol) from Amersham (Amersham, UK)
using a commercial random-priming kit purchased from Pharmacia LKB
(Uppsala, Sweden). Oligonucleotides were synthesized by the
phosphoramidite method in an Applied Biosystems DNA synthesizer (model
392A) and used directly after synthesis. Synthetic peptides
Z-Phe-Arg-AMC, Z-Arg-Arg-AMC, and Z-Arg-AMC were from Bachem
(Bubendorf, Switzerland), and proteinase inhibitor
E-641 was from Sigma.
Screening of a Human Brain cDNA Library--
A computer
search of the GenBankTM data base for sequences with
homology to human cysteine proteinases led us to identify a sequence
derived from an ovarian tumor cDNA clone
(AA283747)2 and showing a
significant similarity with sequences previously determined for other
cathepsins. After looking for additional human expressed sequence tags
(ESTs) similar to this one, we found approximately 20 overlapping ESTs,
spanning around 1350 bp, and useful to design a specific probe for the
gene encoding this putative novel cysteine proteinase. This DNA
fragment was obtained by PCR amplification of cDNA from a
commercially available human brain cDNA library as follows: total
-phage DNA from this brain cDNA library was screened for the
presence of the hypothetical cysteine proteinase using two specific
primers 5'-CACCCGGAACCAGCACAT (primer Z1) and 5'-CGATGGGGTCCCCAATG
(primer Z2) derived from the overlapping ESTs. The PCR reaction was
performed in a GeneAmp 2400 PCR system from Perkin-Elmer for 35 cycles
of denaturation (94 °C, 15 s), annealing (56 °C, 15 s),
and extension (72 °C, 1 min). The PCR product, 708 bp long, was
phosphorylated with T4 polynucleotide kinase and cloned into an
EcoRV-cut pBS vector. The cloned cDNA was sequenced and
found to be identical with the expected sequence. This cDNA was
then excised from the vector, radiolabeled, and used to screen a human
brain cDNA library, according to standard procedures (33).
Hybridization to the radiolabeled probe was carried out for 18 h
in 6 × SSC (1× = 150 mM NaCl, 15 mM
sodium citrate, pH 7.0), 5 × Denhardt's (1× = 0.02% bovine
serum albumin, 0.02% polyvinylpyrrolidone, 0.02% Ficoll), 0.1% SDS,
and 100 µg/ml denatured herring sperm DNA at 55 °C. The membranes
were washed twice for 1 h at 55 °C in 2 × SSC, 0.1% SDS
and exposed to XAR-5 film (Kodak) at
70 °C with intensifying
screens. Following plaque purification, several cloned inserts were
excised by EcoRI digestion and the resulting fragments
subcloned into the EcoRI site of pUC18.
Nucleotide Sequencing-- Selected DNA fragments were inserted in the polylinker region of phage vector M13mp19 and sequenced by the Sanger method (34), using either M13 universal primer or cDNA specific primers and the Sequenase Version 2.0 kit (U. S. Biochemicals). Sequence ambiguities were solved by substituting dITP for dGTP in the sequencing reactions. All nucleotides were identified in both strands. Computer analysis of DNA and protein sequences was performed with the GCG software package of the University of Wisconsin Genetics Computer Group (35).
Fluorescent in Situ Hybridization-- A high density gridded human P1 artificial chromosome (PAC) genomic library (kindly supplied by the Human Genome Mapping Resource Center) was screened by filter hybridization with the full-length cathepsin Z cDNA as probe. Three independent clones were identified enclosing the cathepsin Z gene as demonstrated by PCR and Southern blot analysis. DNA from one of these PAC clones was obtained with the standard alkaline lysis method and then used for fluorescent in situ hybridization mapping. To do that, 2 µg of the PAC DNA was nick translated with biotin-16-dUTP and hybridized to normal male metaphase chromosomes obtained from phytohemagglutinin-stimulated cultured lymphocytes, as described previously (36). Biotinylated probe was detected using two avidin-fluorescein layers. Chromosomes were diamidine-2-phenylindole dihydrochloride-banded, and images were captured in a Zeiss axiophot fluorescent microscope equipped with a CCD camera (Photometrics).
Expression in E. coli--
To prepare an expression vector
suitable for production of recombinant cathepsin Z in E. coli, we first generated a 727-bp DNA fragment containing the
coding sequence for the mature human cathepsin Z by PCR amplification
of the isolated full-length cDNA with primers
5'-ATGCTGCCCAAGAGCTGGGAC and 5'-CGATGGGGTCCCCAAATG. The PCR reaction
was carried out for 30 cycles of denaturation (95 °C, 30 s),
annealing (56 °C, 30 s), and extension (72 °C, 1 min) using
the ExpandTM Long Template PCR System (Boehringer Mannheim)
to reduce error frequency. The PCR product was phosphorylated with T4
polynucleotide kinase, repared with Klenow fragment, and ligated to the
expression vector pGEX-3X (Pharmacia LKB), previously treated with
SmaI and alkaline phosphatase. The resulting plasmid, called
pGEX-3X CathZ, was transformed into E. coli strain
BL21(DE3), and the transformed cells were grown in LB broth containing
100 µg/ml ampicillin at 37 °C for about 16 h, diluted 1/100
with the same medium, and grown to an A600 of
1.0. Then, isopropyl-1-thio--D-galactopyranoside was
added to a final concentration of 1 mM, and the incubation was continued for 3 h. Cells were collected by centrifugation, washed, and resuspended in 0.05 volumes of phosphate-buffered saline,
lysed by using a French press, and centrifuged at 20,000 × g for 20 min at 4 °C. The soluble extract was treated
with glutathione-Sepharose 4B and eluted with glutathione elution
buffer (10 mM reduced glutathione in 50 mM
Tris-HCl, pH 8.0) following manufacturer's instructions.
Enzyme Activity Assays-- The enzymatic activity of purified cathepsin Z produced in E. coli was measured using 20 µM Z-Phe-Arg-AMC, Z-Arg-Arg-AMC, or Z-Arg-AMC as substrates and following the procedure described by Barrett and Kirschke (37) with minor modifications. Assays were performed at 30 °C, in 100 mM sodium acetate buffer, pH 5.5, containing 8 mM dithiothreitol, 2 mM EDTA, and 0.05% Brij 35. Substrate hydrolysis was monitored in a Cytofluor 2350 fluorometer (Millipore) at excitation and emission wavelengths of 360 and 460 nm, respectively. For inhibition assays, the reaction mixture was preincubated with 20 µM E-64 at 30 °C for 15 min, and the remaining activity was determined using the fluorogenic substrate Z-Phe-Arg-AMC as above.
Northern Blot Analysis-- Northern blots containing 2 µg of poly(A)+ RNA of different human tissue specimens and cancer cell lines or 20 µg of total RNA from diverse tumor tissues were prehybridized at 42 °C for 3 h in 50% formamide, 5 × SSPE (1 × = 150 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 7.4), 10 × Denhardt's, 2% SDS, and 100 µg/ml denatured herring sperm DNA. After prehybridization, filters were hybridized with the 708-bp radiolabeled probe corresponding to the core region of the cathepsin Z cDNA. After hybridization, filters were washed with 0.1 × SSC, 0.1% SDS for 2 h at 50 °C and exposed to autoradiography. RNA integrity and equal loading were assessed by hybridization with an actin probe.
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RESULTS |
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Isolation and Characterization of a Human Brain cDNA Encoding a
Novel Member of the Papain Family of Cysteine Proteinases--
In an
attempt to identify novel members of the papain family of cysteine
proteinases, we carried out a computer search of the human EST data
base, looking for sequences with similarity to previously described
family members. This analysis led to the identification of a series of
overlapping ESTs that, after assembly, encoded a protein sequence with
a significant degree of similarity to the C-terminal region of the
different cysteine proteinases of the papain family characterized to
date. A cDNA containing part of the nucleotide sequence
corresponding to these assembled ESTs was obtained by PCR amplification
of total -phage DNA prepared from a commercially available human
brain cDNA library. The amplified DNA fragment (708 bp) was cloned,
and its identity was confirmed by nucleotide sequence analysis. The
cloned fragment was then radiolabeled and used as a probe to hybridize
the same human brain cDNA library utilized for the previous PCR
amplification experiment. Upon screening of approximately 1 × 106 plaque-forming units, a total of four independent
clones showing positive hybridization with the probe were
identified. Then, DNA was isolated from each of these positive
clones, and their nucleotide sequence was determined by standard
procedures. Analysis of the resulting sequences revealed that all of
them seemed to derive from the same gene, which would encode a putative
novel human cathepsin. However, none of these isolated cDNA clones
contained in their nucleotide sequence the coding information
corresponding to the N-terminal region of this novel human enzyme. To
obtain this sequence, we screened a human prostate cDNA library
using as a probe a 85-bp DNA fragment, prepared by PCR amplification and corresponding to the most 5'-region present in the isolated brain
cDNA clones. Upon screening of approximately 1 × 106 plaque-forming units, DNA was isolated from a single
clone showing positive hybridization with the 85-bp probe, and its
nucleotide sequence was determined. Sequence analysis of this positive
clone allowed us to extend by approximately 300 bp the 5'-sequence
determined for the brain cDNA clones as well as to confirm the
remaining part of the nucleotide sequence derived from these clones.
Computer analysis of the 5'-extended sequence revealed an open reading frame 909 bp long, starting with an ATG codon at position 241 and
ending with a TAA codon at position 1150 (Fig.
1). Assuming that translation starts at
this first ATG, the identified open reading frame codes for a protein
of 303 amino acids and a predicted molecular weight of 33,881.
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Chromosomal Mapping of the Human Cathepsin Z Gene-- To establish the chromosomal localization of the human cathepsin Z gene, we first isolated PAC clones containing this gene by screening a genomic library with the full-length cDNA as probe. DNA from one of the isolated clones was then employed in fluorescent in situ hybridization experiments using human male chromosome metaphases. After diamidine-2-phenylindole dihydrochloride banding of the metaphase cells with specific hybridization signals (more than 90% of the cells), fluorescent yellow signals corresponding to the biotinylated PAC clone were clearly mapped to the telomeric region of chromosome 20, in the q13 region (Fig. 3). As can be also seen in Fig. 3, no other chromosome site was labeled above background. A number of genes have been already mapped to this region, including those encoding adenosine deaminase, prostacyclin synthase, or phosphoenolpyruvate carboxykinase (42-44). However, no cysteine proteinase genes of any of the different subfamilies have been previously found to map at this chromosome site.
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Production of Recombinant Cathepsin Z in E. coli and Analysis of
Its Enzymatic Activity--
To elucidate whether the isolated
cathepsin Z cDNA codes for a functional cysteine proteinase, we
expressed the cloned cDNA in a bacterial system following the
strategy described for other cysteine proteinases (45). To do that, we
first prepared by PCR amplification a 727-bp fragment containing the
entire coding sequence for the predicted mature cathepsin Z. After
confirming the nucleotide sequence of the amplified fragment, it was
inserted in the polylinker region of the expression vector pGEX-3X. The resulting plasmid, called pGEX-3X CathZ, as well as the original vector, were transformed into E. coli BL21(DE3), and the
transformed bacteria were induced with
isopropyl-1-thio--D-galactopyranoside. SDS-PAGE analysis
of protein extracts prepared from the induced bacteria revealed that
the bacteria transformed with the recombinant plasmid contained a
fusion protein of about 52 kDa, which was not present in the control
extracts (Fig. 4A). By
contrast, a 29-kDa band corresponding to the parental glutathione
S-transferase was detected in the control extracts but not
in those prepared from the recombinant bacteria (Fig. 4A).
To purify the recombinant cathepsin Z, we performed an affinity
chromatography in a glutathione-Sepharose 4B column, which was eluted
with a reduced glutathione-containing buffer. After elution and
SDS-PAGE analysis of the protein material present in the
chromatographic eluate, a single band of the expected size was detected
(Fig. 4A).
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Expression of Human Cathepsin Z in Tissues and Cancer Cell Lines-- To investigate the presence of cathepsin Z mRNA transcripts in human tissues, Northern blots containing poly(A)+ RNAs extracted from a variety of tissues, including leukocytes, colon, small intestine, ovary, testis, prostate, thymus, spleen, pancreas, kidney, skeletal muscle, liver, lung, placenta, brain, and heart, were hybridized with a 270-bp FokI/EcoRI probe specific for human cathepsin Z. This probe is derived from the 3'-untranslated region of the isolated cDNA for this enzyme, which is the region showing the lowest percentage of identities with other family members (less than 20% identities), thus avoiding the possibility of cross-hybridization signals. After hybridization with the radiolabeled cathepsin Z probe, a single transcript of about 1.7 kb was observed in all examined tissues (Fig. 5A). This widespread distribution of cathepsin Z should be consistent with the possibility that it is a lysosomal enzyme involved in the intracellular protein degradation that takes place in all cell types.
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DISCUSSION |
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In this work we report the identification of a novel human cysteine proteinase called cathepsin Z. We also provide a structural and functional analysis of this novel enzyme and describe its pattern of expression in normal and tumor tissues. Finally, we establish the chromosomal location of the cathepsin Z gene in the human genome, with the finding that it maps to chromosome 20, which is a unique location for any human cysteine proteinase mapped to date.
The identification of cathepsin Z was initially based on a search of the human EST data base looking for sequences with similarity to previously characterized cysteine proteinases. The identified sequences were then PCR-amplified and used to screen cDNA libraries from different human tissues. This strategy led finally to the isolation of a full-length cDNA coding for a protein with sequence similarity to human cysteine proteinases of the papain family, which was tentatively called cathepsin Z. The identified sequence for this novel enzyme exhibits the domain structure characteristic of cysteine proteinases, including a signal sequence, a prodomain, and a catalytic domain responsible for the proteolytic activity of these proteins (26). It also contains a series of residues proposed to be important in the catalytic mechanism of these enzymes such as the active site Cys residue which is transiently acetylated during peptide hydrolysis, as well as the Asn and His residues which form the catalytic triad of cysteine proteinases (39-41). However, a more detailed structural analysis of the sequence determined for cathepsin Z revealed some specific features for this novel enzyme. Thus, this novel cysteine proteinase contains three peptide insertions unique among all family members (Fig. 2). The first of these insertions consists of the introduction of the tripeptide (His-Ile-Pro) immediately adjacent to the Gln residue of the oxyanion hole. This insertion is also present in the murine homologue of cathepsin Z3, and would considerably increase the short distance (5 residues) separating this Gln residue from the active site Cys of the remaining cysteine proteinases of the papain family. Therefore, it is tempting to speculate that it could confer distinct catalytic properties or affect the substrate specificity of cathepsin Z. The two other insertions consist of an 8-residue peptide located in the middle of the molecule and a 14-residue peptide in the C-terminal end of the molecule, but their putative structural and functional significance on cathepsin Z properties remains unknown. Nevertheless, the most striking feature of the sequence of cathepsin Z is its unusually short propeptide when compared with those present in other cysteine proteinases. It is well known that all cysteine proteinases are synthesized as inactive precursors with an N-terminal propeptide which acts as an intrinsic inhibitor of the proteolytic activity (48). The propeptide has also found to be essential for correct folding of some cysteine proteinases and for their stabilization upon exposure to changes in pH environments (49, 50). The overall sequence similarities among the propeptides of the different cysteine proteinases are low, but according to structural features, they can be assigned to two groups (51, 52). The first group contains cathepsin L-like enzymes with proregions greater than 90 amino acids in length and two highly conserved motifs called ERFNIN and GNFD. The second group comprises the cathepsins B from different sources and is characterized by a smaller proregion of about 60 amino acids lacking the ERFNIN motif. However, the proregion of cathepsin Z cannot be assigned to any of these groups because it is only 41 residues in length and lacks both conserved domains. Furthermore, this propeptide sequence does not contain any lysine residue, despite the fact that lysine-based structures present in the proregion of different cathepsins have been proposed to act as the recognition sites for the mannose phosphorylation required for intracellular targeting of these proteins (53). Taking together these structural data, it seems clear that the proregion of cathepsin Z markedly deviates from those of all previously characterized family members, suggesting that it could be a member of a different subfamily of enzymes. Consistent with this hypothesis, chromosomal location of the cathepsin Z gene has revealed that it maps to chromosome 20. This position differs from those reported for the remaining cysteine proteinase genes of the papain family (54-60), providing additional support to the above structural data that suggest that this novel cysteine proteinase is not closely related to the other members of the papain family. In addition to its possible value in the context of evolutionary studies of the human cysteine proteinases, knowledge of the chromosomal location of the cathepsin Z gene may be useful in searching for putative diseases related to abnormalities in this protein, as already demonstrated for cathepsin K (61).
In this work we have also provided evidence that cathepsin Z is expressed in all normal tissues analyzed, which suggests a putative general role of this protein as a proteolytic enzyme involved in the normal intracellular protein turnover taking place in all cell types. This housekeeping role of cathepsin Z in human tissues would be similar to that proposed for cathepsins B, L, H, and O, but distinguishes this enzyme from a series of recently described cysteine proteinases which appear to play highly specific roles in those tissues in which they are overexpressed or even exclusively expressed. This is the case of cathepsins K, S, W, and L2, predominantly expressed in osteoclasts, lymphatic tissues, T-lymphocytes, and thymus and testis, respectively, and proposed to be involved in bone remodeling (cathepsin K), antigen presentation (cathepsin S), regulation of T-cell cytolytic activity (cathepsin W), and regulation of the immune response and fertilization processes (cathepsin L2) (20, 21, 61, 62). The expression analysis of cathepsin Z also revealed a ubiquitous presence of this enzyme in a series of human cancer cell lines and primary tumors from different sources. This finding suggests that cathepsin Z may be somewhat linked to the malignant transformation of human cells as already shown for other cysteine proteinases (8, 47) and adds a new interest to the study of this novel enzyme.
In summary, according to the results of this work, human cathepsin Z is a novel member of the papain family of cysteine proteinases that shows clear differences with the remaining family members characterized to date. The occurrence of a series of unique features in its structure, including diverse peptide insertions and an unusually short propeptide region, together with its chromosomal location at 20q13, distinguishes this enzyme from other cysteine proteinases and suggests that it may be the first representative of a new cathepsin subfamily. The availability of recombinant cathepsin Z and specific reagents for this proteinase will be very helpful in evaluating the functional significance of these structural differences as well as in studying the potential role of this novel enzyme in the protein degradative processes occurring in normal and pathological conditions, including cancer.
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Balbín and J. P. Freije for helpful comments and S. Alvarez for excellent technical assistance.
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FOOTNOTES |
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* This work was supported by Grant SAF97-0258 from the Comisión Interministerial de Ciencia y Tecnología, Grant BMH4-CT96-0017 from EU-BIOMED II, and grants from Glaxo-Wellcome, Spain.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF032906.
Recipient of a fellowship from Ministerio de Educación y
Ciencia (Spain).
¶ To whom correspondence should be addressed: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Tel.: 34-85-104201; Fax: 34-85-103564 or 34-85-232255; E-mail: CLO{at}DWARF1.QUIMICA.UNIOVI.ES.
1 The abbreviations used are: E-64, trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane; bp, base pair(s); kb, kilobase(s); EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; PAC, P1 artificial chromosome; Z-Phe-Arg-AMC, benzyloxycarbonyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarin.
2 Deposited by L. Hillier, N. Clark, T. Dubuque, K. Elliston, M. Hawkins, M. Holman, M. Hultman, T. Kucaba, M. Le, G. Lennon, M. Marra, J. Parsons, L. Rifkin, T. Rohlfing, F. Tan, E. Trevaskis, R. Waterston, A. Williamson, P. Wohldmann, and R. Wilson, WashU-Merck EST project.
3 I. Santamaría and C. López-Otín, unpublished data.
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REFERENCES |
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