Nucleic Acids Research, Vol. 18, No. 22 6703
.=) 1990 Oxford University Press
Nucleotide sequence of a cDNA for porcine collagenase, obtained by PCR
type
I
N.J.Clarke, M.C.O'Harel, T.E.Cawston' and G.P.Harper SmithKline Beecham Pharmaceuticals, Coldharbour Road, The Pinnacles, Harlow, Essex CM19 5AD and 'Rheumatology Research Unit, Level E6, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK EMBL accession no. X54724
Submitted October 12, 1990
using double-stranded dideoxy methodology. The identity of the clone was confirmed by comparison of the nucleotide sequence data with the amino acid sequence (15 residues) obtained for the N-terminus of the active form of purified porcine type I collagenase (unpublished results) and with the cDNA's encoding the human and rabbit homologues. The position of the N-terminus of the mature enzyme is marked with an asterisk below. The cDNA contains all the coding information for the mature form, and most of that for the pro-peptide of porcine type I collagenase.
Type I collagenase (EC 3.4.24.7) belongs to a family of structurally related neutral metalloproteinases that also includes the stromelysins, type IV collagenases and pump-1 (1). Synthetic oligonucleotide primers were made, corresponding to nucleotides 140-163 (coding strand) and 1454-1475 (non-coding strand) of the cDNA for human fibroblast collagenase (2). Within these regions both the human (2) and rabbit (3) fibroblast collagenase cDNA's have identical nucleotide sequences, compared to the less than 50% homology exhibited by the other cloned members of the family (1). Porcine cDNA (20 ng) from synovial fibroblasts was subjected to PCR amplification using the synthetic oligonucleotide primers described. After 30 cycles, the PCR reaction mixture was purified by agarose gel electrophoresis followed by the GenecleanT procedure. A fragment of approximately 1.3 kb was subcloned into the SmaI site of pUC18 and the insert from several of the resulting clones was sequenced
REFERENCES 1. Docherty,A.J.P. and Murphy,G. (1990),Ann. Rheum. Dis. 49, 469-479. 2. Goldberg,G.I., Wilhelm,S.M., Kronberger,A., Bauer,E.A., Grant,G.A. and
Eisen,A.Z. (1986) J. Biol. Chem. 261, 6600-6605.
3. Fini,M.E., Plucinska,I.M., Mayer,A.S., Gross,R.H. and Brinckerhoff,C.E.
(1987) Biochemistry 26, 6156-6165.
80
1 AGAAACACAAGAGCAAGATGTGGAGATAGTGCAGAAATACCT E
T
E Q D
Q
E
V
I
Q K
V
K
L
Y
N
Y
Y
L
N
N
G
D
S
V
P
V
81 TGGAAAAGAAAAGGAACAGTGGCCTCGTAGTTGAAAAACTAAAGCAAATGCAACAATTCTTTGGGCTGAAAGTAACGGGG 160 E K K R N S G L V V E K L K Q M Q Q F F G L K V T G
161 AAACCAGACGCTGAAACTCTGAATGTGATGAAGCAGCCCAGATGTGGAGTTCCTGACGTGGCTGAGTTTGTTCTCACTCC 240 D
P
K
A
E
V
N
L
T
K Q
M
P
G
C
R
P
V
V
D
E
A
L
V
F
P
T
-
320
241 G
P
N
W E
R
H
T
N
Y
T
L
I
R
T
Y
N
E
L
D
P
R
S
V
D
E
D
321 ACCGTGCCATTGAGAAAGCCTTTCAACTCTGGAGCAATGTCTCACCCTTGACCTTCACCAAGGTCTCCGAGGGTCAAGCA 400 A
R
E
I
K
A
Q
F
W
L
S
V
N
P
S
L
T
F
T
E
S
V
K
Q A
G
-
401 GACATAATGATATCCTTTGTCAGGGGAGATCATCGTGACAATTCTCCCTTTGATGGACCTGGAGGAAACCTTGCTCATGC 480 D I M I S F V R G D H R D N S P F D G P G G N L A H A -
481 TTTTCAGCCAGGCCCAGGTATTGGAGGGGATGCTCATTTTGATGAAGATGAAAGGTGGACCAAAAATTTCAGAGATTATA 560 Q
F
P
P
G
G
I
G
D
G
A
H
F
E
D
R W
E
D
K
T
F
N
R
Y
D
N -
561 ACTTGTATCGTGTGGCTGCTCATGAACTGGGCCATTCCCTTGGCCTTTCTCATTCCACTGACATTGGGGCTTTGATGTAC 640 L
R
Y
A
V
H
A
E
L
H
G
L
S
G
S
L
S
H
I
D
T
G
A
Y
M
L
-
641 CCCAACTACATCTACACTGGTGATGTTCAGCTGTCCCAAGATGACATTGATGGCATCCAGGCCATCTATGGACCTTCCGA 720 P
Y
N
I
Y
T
G
Q
V
D
S Q
L
D
D
D
I
G
A
Q
I
P
G
Y
I
S
E
721 AAACCCCGTTCAGCCAAGTGGCCCACAAACCCCACAAGTGTGTGATAGTAAGCTAACTTTTGATGCTATAACTACACTTC 800 N P V Q P S G P Q T P Q V C D S K L T F D A I T T L R 801 GGGGAGAACTAATGTTCTTTAAAGACAGGTTCTACATGCGCACAAATTCCTTCTACCCTGAAGTGGAGCTCAATTTCATT 880 G
E
L
M
F
F
K
D
R
F
Y
M
R
T
S
N
F
Y
E
P
V
L
E
N
F
I
-
881 TCTGTGTTCTGGCCACAAGTGCCAAATGGACTTCAAGCTGCTTATGAGATTGCCGATAGAGATGAAGTCCGGTTTTTCAA 960 S V F W P Q V P N G L Q A A Y E I A D R D E V R F F K 961 AGGTAACAAGTACTGGGCTGTTAGGGGGCAGGATGTGCTATACGGATACCCCAAGGACATCCACAGATCCTTTGGCTTCC 1040 G N K Y W A V R G Q D V L Y G Y P K D I H R S F G F P 1041 CTAGTACTGTGAAGAATATCGATGCTGCTGTTTTTGAGGAAGATACTGGGAAAACATACTTCTTTGTTGCTCACGAATGC 1120 S T V K N I D A A V F E E D T G K T Y F F V A H E C
1121 TGGAGGTATGATGAATATAAACAATCCATGGATACAGGTTATCCCAAAATGATAGCAGAAGAATTTCCTGGGATTGGCAA 1200 W R Y D E Y K Q S M D T G Y P K M I A E E F P G I G N 1201
1280 T -
CAAAGTTGATGCTGTTTTCCAGAAAGACGGATTTCTATATTTCTTCCATGGAACAAGGCAGTACCAATTTGATTTCAAAA A
D
V
K
D
F Q K
V
Y
L
F
G
F
F
H
G
R
T
Q
1281 CGAAGAGAATTTTGACTCTCCAAAAAGCTAATAGCTGGTTCAACTGCAGGAAAAAT 1336 K
R
I
L
T
L
Q
K
A
N
S
W
F
N
C
R
K
N
-
Y
Q
F
D
F
K
.=) 1990 Oxford University Press
Nucleotide sequence of a cDNA for porcine collagenase, obtained by PCR
type
I
N.J.Clarke, M.C.O'Harel, T.E.Cawston' and G.P.Harper SmithKline Beecham Pharmaceuticals, Coldharbour Road, The Pinnacles, Harlow, Essex CM19 5AD and 'Rheumatology Research Unit, Level E6, Addenbrookes Hospital, Hills Road, Cambridge CB2 2QQ, UK EMBL accession no. X54724
Submitted October 12, 1990
using double-stranded dideoxy methodology. The identity of the clone was confirmed by comparison of the nucleotide sequence data with the amino acid sequence (15 residues) obtained for the N-terminus of the active form of purified porcine type I collagenase (unpublished results) and with the cDNA's encoding the human and rabbit homologues. The position of the N-terminus of the mature enzyme is marked with an asterisk below. The cDNA contains all the coding information for the mature form, and most of that for the pro-peptide of porcine type I collagenase.
Type I collagenase (EC 3.4.24.7) belongs to a family of structurally related neutral metalloproteinases that also includes the stromelysins, type IV collagenases and pump-1 (1). Synthetic oligonucleotide primers were made, corresponding to nucleotides 140-163 (coding strand) and 1454-1475 (non-coding strand) of the cDNA for human fibroblast collagenase (2). Within these regions both the human (2) and rabbit (3) fibroblast collagenase cDNA's have identical nucleotide sequences, compared to the less than 50% homology exhibited by the other cloned members of the family (1). Porcine cDNA (20 ng) from synovial fibroblasts was subjected to PCR amplification using the synthetic oligonucleotide primers described. After 30 cycles, the PCR reaction mixture was purified by agarose gel electrophoresis followed by the GenecleanT procedure. A fragment of approximately 1.3 kb was subcloned into the SmaI site of pUC18 and the insert from several of the resulting clones was sequenced
REFERENCES 1. Docherty,A.J.P. and Murphy,G. (1990),Ann. Rheum. Dis. 49, 469-479. 2. Goldberg,G.I., Wilhelm,S.M., Kronberger,A., Bauer,E.A., Grant,G.A. and
Eisen,A.Z. (1986) J. Biol. Chem. 261, 6600-6605.
3. Fini,M.E., Plucinska,I.M., Mayer,A.S., Gross,R.H. and Brinckerhoff,C.E.
(1987) Biochemistry 26, 6156-6165.
80
1 AGAAACACAAGAGCAAGATGTGGAGATAGTGCAGAAATACCT E
T
E Q D
Q
E
V
I
Q K
V
K
L
Y
N
Y
Y
L
N
N
G
D
S
V
P
V
81 TGGAAAAGAAAAGGAACAGTGGCCTCGTAGTTGAAAAACTAAAGCAAATGCAACAATTCTTTGGGCTGAAAGTAACGGGG 160 E K K R N S G L V V E K L K Q M Q Q F F G L K V T G
161 AAACCAGACGCTGAAACTCTGAATGTGATGAAGCAGCCCAGATGTGGAGTTCCTGACGTGGCTGAGTTTGTTCTCACTCC 240 D
P
K
A
E
V
N
L
T
K Q
M
P
G
C
R
P
V
V
D
E
A
L
V
F
P
T
-
320
241 G
P
N
W E
R
H
T
N
Y
T
L
I
R
T
Y
N
E
L
D
P
R
S
V
D
E
D
321 ACCGTGCCATTGAGAAAGCCTTTCAACTCTGGAGCAATGTCTCACCCTTGACCTTCACCAAGGTCTCCGAGGGTCAAGCA 400 A
R
E
I
K
A
Q
F
W
L
S
V
N
P
S
L
T
F
T
E
S
V
K
Q A
G
-
401 GACATAATGATATCCTTTGTCAGGGGAGATCATCGTGACAATTCTCCCTTTGATGGACCTGGAGGAAACCTTGCTCATGC 480 D I M I S F V R G D H R D N S P F D G P G G N L A H A -
481 TTTTCAGCCAGGCCCAGGTATTGGAGGGGATGCTCATTTTGATGAAGATGAAAGGTGGACCAAAAATTTCAGAGATTATA 560 Q
F
P
P
G
G
I
G
D
G
A
H
F
E
D
R W
E
D
K
T
F
N
R
Y
D
N -
561 ACTTGTATCGTGTGGCTGCTCATGAACTGGGCCATTCCCTTGGCCTTTCTCATTCCACTGACATTGGGGCTTTGATGTAC 640 L
R
Y
A
V
H
A
E
L
H
G
L
S
G
S
L
S
H
I
D
T
G
A
Y
M
L
-
641 CCCAACTACATCTACACTGGTGATGTTCAGCTGTCCCAAGATGACATTGATGGCATCCAGGCCATCTATGGACCTTCCGA 720 P
Y
N
I
Y
T
G
Q
V
D
S Q
L
D
D
D
I
G
A
Q
I
P
G
Y
I
S
E
721 AAACCCCGTTCAGCCAAGTGGCCCACAAACCCCACAAGTGTGTGATAGTAAGCTAACTTTTGATGCTATAACTACACTTC 800 N P V Q P S G P Q T P Q V C D S K L T F D A I T T L R 801 GGGGAGAACTAATGTTCTTTAAAGACAGGTTCTACATGCGCACAAATTCCTTCTACCCTGAAGTGGAGCTCAATTTCATT 880 G
E
L
M
F
F
K
D
R
F
Y
M
R
T
S
N
F
Y
E
P
V
L
E
N
F
I
-
881 TCTGTGTTCTGGCCACAAGTGCCAAATGGACTTCAAGCTGCTTATGAGATTGCCGATAGAGATGAAGTCCGGTTTTTCAA 960 S V F W P Q V P N G L Q A A Y E I A D R D E V R F F K 961 AGGTAACAAGTACTGGGCTGTTAGGGGGCAGGATGTGCTATACGGATACCCCAAGGACATCCACAGATCCTTTGGCTTCC 1040 G N K Y W A V R G Q D V L Y G Y P K D I H R S F G F P 1041 CTAGTACTGTGAAGAATATCGATGCTGCTGTTTTTGAGGAAGATACTGGGAAAACATACTTCTTTGTTGCTCACGAATGC 1120 S T V K N I D A A V F E E D T G K T Y F F V A H E C
1121 TGGAGGTATGATGAATATAAACAATCCATGGATACAGGTTATCCCAAAATGATAGCAGAAGAATTTCCTGGGATTGGCAA 1200 W R Y D E Y K Q S M D T G Y P K M I A E E F P G I G N 1201
1280 T -
CAAAGTTGATGCTGTTTTCCAGAAAGACGGATTTCTATATTTCTTCCATGGAACAAGGCAGTACCAATTTGATTTCAAAA A
D
V
K
D
F Q K
V
Y
L
F
G
F
F
H
G
R
T
Q
1281 CGAAGAGAATTTTGACTCTCCAAAAAGCTAATAGCTGGTTCAACTGCAGGAAAAAT 1336 K
R
I
L
T
L
Q
K
A
N
S
W
F
N
C
R
K
N
-
Y
Q
F
D
F
K
