Molecular cloning and expression of human ribonuclease 4 cDNA

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Biochimica et Biophysica Acta 1261 (1995) 424-426

Biochi ~mic~a et BiophysicaA~ta

Short Sequence-Paper

Molecular cloning and expression of human ribonuclease 4 cDNA " Masaharu Seno *, Jun-ichiro Futami, Yoshiaki Tsushima, Kazumi Akutagawa, Megumi Kosaka, Hiroko Tada, Hidenori Yamada Department of BioengineeringScience, Faculty of Engineering, Okayama University, Tsushima-Naka, Okayama 700, Japan Received 9 December 1994; revised 1 February 1995; accepted 2 February 1995

Abstract

A cDNA coding for human ribonuclease 4 was isolated from a pancreas cDNA library and sequenced. This cDNA (996 bp) includes an entire open reading frame encoding mature protein (119 aa) following signal peptide (28 aa). Expression of mature protein in Escherichia coli showed an apparent molecular mass of about 16 kDa, which was slightly lower than the mature form of human RNase 1, in SDS-PAGE. Keywords: Pancreatic type ribonuclease family; RNase 4; cDNA cloning; Gene expression; (Pancreas); (Human)

Pancreatic type ribonucleases (RNases) constitute a large superfamily crossing over species [1]. Bovine pancreatic RNase (RNase 1), largely known as RNase A, is classical and the first and best characterized. Members of this RNase family derived from amphibian, bovine and human have recently been found and characterized. Especially amphibian RNases, Onconase [2] and lectins [3], bovine seminal RNase [4] and human angiogenin (RNase 5) [5] appear to play important roles as factors controlling cellular growth and development. In human, the counterparts of neither Onconase, amphibian lectins nor seminal RNase have been identified, while five homologues are found in various tissues. RNase 5 (angiogenin), which induces neovascularization on chicken embryo chorioallantoic membrane and rat cornea, is a unique homologue of RNase 1 with a ribonucleolytic activity markedly different from RNase 1 [6]. Another two members of the family, RNase 2 and 3, were originally isolated as neurotoxin from eosinophil and named eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), respectively [7]. Very recent study has revealed the existence of a novel member of RNase, which specifically cleaves on the 3'

~ The nucleotide sequence data reported in this paper have been submitted to the GSDB, DDBJ, EMBL and NCBI nucleotide sequence databases under the accession number D37931. * Corresponding author. Fax: + 81 86 2535755. 0167-4781/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved

SSDI 016 7 - 4 7 8 1 ( 9 5 ) 0 0 0 4 0 - 2

side of uridine, called RNase 4 [8]. RNase 4 was originally isolated from the conditioned medium of human colon adenocarcinoma HT-29 [9], which was also the original source of RNase 5, and plasma [8], while the counterparts of RNase 4 in bovine and porcine were purified from liver [10,11] implying widespread distribution of RNase 4. To make clear the biological significance of heterogeneity of pancreatic type RNases, cDNA cloning for RNase 4 was carried out by screening cDNA library prepared from human pancreas [12] and a bacterial expression system was established. First we designed three oligonucleotide primers, two forward and one reverse primer, from the primary structure of human RNase 4 [8]. One of the forward primers was a mix of 128 oligonucleotides of 20-mer with the sequence of 5 ' - C A A / G G A C / T G G A / C / G / T A T G T A C / T C A A / GA/CG-3' (#1) reverse-translated from Q1-R7 and the other a mix of 48 oligonucleotides of 20-mer with the sequence of 5 ' - T A C / T T G C / T A A C / T C / T I ' A / C / G / TATGATGCA-Y (#2) from y24_Q30. The reverse primer was designed as a mix of 32 oligonucleotides of 20-mer with the sequence of 5 ' - C C C / T I ' C A / G T G A / G C A A / GTTCATC/TTT-3' (#3), which was complementary to the one from K 6 8 - Q 74. Polymerase chain reaction on the phage solution of cDNA library using the pair of primers #1 and # 3 showed an amplified DNA fragment of 200 bp, which could be a template for amplification of DNA fragment of 130 bp with the pair of primers # 2 and #3.

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M. Seno et al. /Biochimica et Biophysica Acta 1261 (1995) 424-426 "L0

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GAATTCCGGCCCCCCTCTAAGATAC

TGATGGCTCTGCAGAGGAC C CATTCATTGCTTCTGCTTTTGCTGCTGACCCTGCTGGGGCTGGGG /4 A L Q R T H S L L L L L L L T L L G L -28 -20 -10 CTGGTCCAGCCCTCCTATGGC CAGGATGGCATGTACCAGCGATT CCTGCGGCAACACGTGCACCCTGAGGAGACAGGTGGCAGTGATCGC L V Q P S Y G Q D G M • Q R F L l Q H V H P E E T G G S D -1

1

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90 G 180 R

20

TACTGCAACTTGATGATGCAAAGAC GGAAGATGACTTTGTATCAC TGCAAGCGCTTCAACACCTTCATCCATGAAGATATCTGGAACATT Y

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N

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M

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Q 30

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R

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F

N

T

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270

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C GTAGTATCTGCAGCAC CAC CAATATC CAATGCAAGAAC GGCAAGAT GAACTGCCATGAGGGTGTAGTGAAGGTCACAGATTGCAGGGAC It

S

I

C

S

T

T N I Q C K N G K M II C H E G V V K V T D C R 60 70 80 ACAGGAAGTTCCAGGGCA•CCAA•TGCAGATAT•GGGCCATAGcGAGCA•TAGACGTGTTGTCATTGCCTGTGAGGGTAAC•CACAGGTG T G 8 8 R ~ P N C R l R A I A B T R R V V I A C E G N P Q 90 100 110

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CACCATGTAGGGATTATCGCGAGTGGTTGACCTTACACTTACTCCTTAAATAGCAGTGAGTAATG

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CATTTGAGC TGTCCCAGGCTCTGTC TCCTCAGCTCATTTCCTACTCTTTTTCTCTATATAACTCATTCTATTAAATACATTGCACCAAAG AGATATGGAGACATAAAccTGTAATGAATGAGGCTGGGCTTTTCTGTAATAAGCTTCCTTTTATAATACTGGTCAGCTTAGTCTCTCAGA TCCTATCCTGTGGAATTTAGTTATTATGTGTATTTATGTAGTATTTCAAACATTTCAAAATGCTTTCATCTATGTTTATCACATTTTAAT ACCACAGAC TTATAATGATGTCACTACATATAGAAGC TCAAAGT TAAGGGATTTGC TGAAGACTGTAAAGTTAATGGAAGAATTGAGACA AAAATCCAGTGTAGCTGGCCACTTATCCAGGGCTTTTTCTACTTCATCACAAGGAATGTTTTGAAAGTGTCTGCTTTTTTT•TCCTTCCG GAATTC

630 720 810 900 990 996

Fig. 1. Nucleotide sequence of human RNase 4 eDNA and the predicted amino acid sequence. The amino acids in signal peptidc are italicized. The sequences derived from EcoRl linker used to construct eDNA library are underlined. The sizes of both D N A fragments were consistent with the length of encoded amino acid sequences and confirmed the expression of RNase 4 gene in pancreas. The library was screened using the D N A fragment of 200 bp as a probe. Screening 105 independent plaques, 4 positively hybridized plaques were obtained. One of the phage clones with the inserted e D N A of around 1 kbp was selected, and the e D N A was subcloned into a phagemid vector pUC119 [13] to construct the phagemid pBO52 and analyzed. The nucleotide sequence analysis revealed the predicted signal sequence consisting of 28 amino acids followed by the primary structure identical to that of mature protein purified from plasma [8] (Fig. 1). The position of putative

initiation of translation was determined by the consensus of signal peptide sequences of the other RNases, which coincided well with the counterparts (Fig. 2). This putative signal sequence composed of 28 amino acid residues shows only one substitution of S - e l to V when compared with that of porcine RNase 4 [14]. Including signal sequence, sequence comparison of human RNase 4 with RNase 1 [12], 2 [10], 3 [11] and 5 [5] shows identity of 46.9, 22.4, 25.9 and 40.8%, respectively (Fig. 2). The sequences of RNase 4s are well conserved between human, bovine and porcine exhibiting about 90% identity [8] in contrast to the fact that the sequence identity of RNase 1 is 71.3% between human and bovine [12]. This highly conserved =

RNase4 RNasel RNase5 RNase2 RNase3

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RNase4 RNasel RNase5 RNase2 RNase3

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RNase4 RNasel RNase5 RNase2

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2= 3 4 4 1 RKMTLYHCKRFNTFIHED IWNIRS ICSTTNI QCKNGKM--NCH--E~RDTGSSRRNMTQGRCKPVNTFVHEPLVDVQNVCFQEKVTCKNGOG-NCYKSNSSMH ITDCRLTNGSRRGLT- SPCKD INTF IHGNKRS IKAI CENKNGNPHRE .... NLRI SKS SFQVTTCKLHGGSP INNYQRRCKNQNTFLLTTFANVVNVCGNPNMTCPSNKTRKNCHHSGSQVPLIHCNLTTPSP~ IN N Y R W R C K N Q N T F L R T T F VVN CGNQS IRCPHNRTLNNCHRSRFRVPLLHCDL INPGAQ

88 91 88 91 91

119 128 123 134 134

Fig. 2. Comparison of amino acid sequences of human RNases. The sequences of RNase 1, 2, 3 and 5 are taken from [12], [10], [11] and [5], respectively. The secretion signals are shown in lower case. The pairs of cysteine residues in each disulfide bridge are shown in identical numbers at the top of the column. Each ' = ' is put on the amino acid residues in the active site. Completely conserved amino acids are shown by ' * ' at the bottom of the column.

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M. Seno et al. / Biochimica et Biophysica Acta 1261 (1995) 424-426

amino acid sequence in evolution implies significance of structure-function relationships of RNase 4. The essential amino acid residues such as histidine and lysine residues in the active site are completely conserved (Fig. 2). The cysteine residues involved in disulfide bridges are also well conserved, however, RNase 5 is only one exception lacking the fourth pair (Fig. 2). Characteristic feature of RNase 4 in compared with the other members of pancreatic RNases is the deletions in the carboxy-terminal sequence. There exists an accurately conserved portion of amino acid sequence, V / L P V H F / L D , which is localized in the carboxy-terminal. Flanking this core sequence, delet i o n s / i n s e r t i o n s of amino acid residues are found in all members of pancreatic type of human RNases. Insertion in the immediate amino terminal side of this core sequence is found in RNase 2 and 3. A n d deletion in the carboxyterminal side of this core sequence is found in RNase 2, 3 and 4. Absence of the sequences in both sides of the core sequence makes the composition of RNase 4 in the minimum number of amino acid residues (119 aa) in the family. Recombinant human RNase 4 was produced in E. coli

kDa

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66.2 45.0 31.0

21.5 14.4 6.5

Fig. 3. SDS-PAGE analyses of human RNase 4 produced in E. coil The signal peptide coding sequence replaced with an ATG codon, human RNase 4 cDNA was modified to code mature protein and ligated downstream of T7 promoter to construct the expression plasmid pBO61. E. coli MM294 lysogenized with DE3 phage harboring pLysS [15] was transformed with pBO61. The transformant thus obtained was cultured and recombinant protein was induced with IPTG as described previously [15]. The extracts prepared from whole bacterial cells cultured in the absence of IPTG (lane 1) and in the presence of IPTG (lane 2) were electrophoresed under reducing conditions and the gel was stained with Coomassie brilliant blue R250. The induced band of protein is shown by an arrowhead. Lane 3, recombinant human RNase 1; lane 4, des. 1-7 hRNase 1; lane 5, bovine RNase A (Sigma, USA). The sizes of molecular mass markers (Bio-Rad, USA) correspond to bovine pancreatic aprotinin (6.5 kDa), hen egg white lysozyme (14.4 kDa), soybean trypsin inhibitor (21.5 kDa), bovine carbonic anhydrase (31 kDa), hen egg white ovalbumin (45 kDa), bovine serum albumin (66.2 kDa).

MM294 lysogenized with DE3 phage under the control of T7 promoter [15]. A band of protein induced with isopropyl-/3-D-thiogalactopyranoside (IPTG) at the molecular mass of approx. 16 kDa was observed in S D S - P A G E under reducing conditions (Fig. 3). This induced band of RNase 4 was electrophoresed at the molecular mass slightly lower than that of mature form of human RNase 1 and at that almost equivalent to those of human RNase 1, whose amino terminal was truncated by 7 amino acid residues, designated as des. 1 - 7 hRNase 1, and bovine RNase 1 (Fig. 3). Analyses of both amino-terminal sequences and carboxy-terminal amino acid residues revealed that mature human RNase 1 and des. 1 - 7 hRNase 1 composed of 129 and 122 amino acid residues as were designed, respectively. The recombinant human RNase 4 is designed to be a mature type of protein composed of 119 amino acid residues with an extra methionine residue for translational initiation at its amino-terminal. These numbers of amino acid residues well elucidate the relative mobility of these RNases in SDS-PAGE. Further characterization including the cytotoxic a n d / o r anti-tumor activity of human RNase 4 is proceeding with this recombinant protein prepared from E. coli. We acknowledge Drs, K. Igarashi, S. Seno and Mr. M. Kita for their continuous encouragement.

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