The Comptete cDNA Coding Sequence for the

Bovine Proc 2(I) Chain of Type I Procoltagen

TOMOKOSHIRAI *~, SHUNJI HATTORIt, MASAHIROSAKAGUCHI*, SAKAEINOUYD,
AKINORI KIMURA*, TETSUYAEBIHARAt, SHINKICHI IRIEt, YUTAKA NAGAI* and
HISAE HORI*
*Division of Adult Diseases, Medical Research Institute, Tokyo Medical and Dental University,
t Nippi Research Institute of Biomatrix,
Department of Immunology and
Infectious Diseases Surveillance Center, National Institute of Infectious Diseases, Tokyo, Japan.

Abstract
The complete sequence of the cDNA for the pro0t2(I) chain of bovine type I procollagen is
presented. The encoded amino acid sequence shows 92.0% identity to the human pro0t2(l)
collagen chain.
Key words: bovine, pro0t2(I) collagen.

Introduction and Results

Collagen is the major component of the extracellular
matrix in multicellular animals. In higher animals, at
least 19 genetically distinct collagen types are known
(Bateman et al., 1996). Type I collagen is rich in bone,
tendon, skin and other connective tissues. The molecule
is a heterotrimer composed of two ctl(I) chains and one
0t2(I) chain. A primary structure of the ctl and t2 chains
of type I collagen was defined by biochemical methods
from several species (Bornstein and Traub, 1979). Type I
collagen is highly conserved compared to other proteins
and therefore has been used as low-antigenic material.
Thus type I collagen and collagen-derived gelatin of
bovine origin are widely used as foods, cosmetics and in-

Present address: Nippi Research Institute of Biomatrix,

Tokyo, 120.
2The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank nucleotide sequence
databases with the accession number AB008683.
Matrix Biology Vol. 17/1998, pp. 85-88
1998 by Gustav Fischer Verlag

gredients of medicines. In recent years, however, it has

been reported that systemic allergic reactions to
Japanese encephalitis vaccines might be caused by a
bovine-derived gelatin employed as a stabilizer of the
vaccine (Sakaguchi et al., 1997). The complete primary
structure is essential for screening the epitope and for
making of low-antigenic collagen and gelatin. A full
amino acid sequence of bovine 0d(I) chain was published except for the signal peptide and C-propeptide regions (Miller, 1984). However, for the bovine 0t2(I)
chain, only partial amino acid sequences have been
available (Bornstein and Traub, 1979), and no complete
primary structure has been reported so far. In this paper,
we report a complete cDNA coding sequence for bovine
0~2(I).
To clone bovine pro(z2(I) cDNA, probe H f - l l 3 1 coding human pro(z2(I) (Bernard et al., 1983) was labelled
with Ic~-32p]dCTP by a multiprime DNA labelling system
and used to screen a bovine ovary cDNA library (Clontech) and a bovine aorta cDNA library (Stratagene). Six
overlapping clones were obtained from these libraries,

Boviae

-110
taagttggaggtactggcca
-g0 cgactgcatgcctgcgccc9ccaggtgatacctccgcc9gtgaccaggggctctgcgcacaaggagtcgcatgtct9agtgtagac
i atgctcagctttgtggatacgcggactttttgtgcttgcagtaacttcgtgcctgaaatgaatccttacaagaggaactgca
I

S C
L

HumAn

Human

P
P

G
T

-L

180
60

181 c t c c t g g c c c c c t g g c c c c t g c c c c c t g g c t g c g a a c t t t c t g c c a g t t t g a t c a a a g a . . . g g t g t
p

264

--

265 g g a c c a t g g g g c t g a g g g a t c c g c c g c t c t 9 a c c t g c c c t c a g g t t c c a g g c t g g g g a g c
8 9 G P M G L M G P R G P P G A S G A P G p Q G F Q G p p
A

88

356
G

]
A

Human

355 ~ g t ~ a a c c ~ g g t c a g a c ~ t c c t g c a g g t g c t c g L g g c c c g c c L g g c c c t c c t g g c a a g g c t g g t g a g ~ a ~ g g t c a c c c t g g a a a a c ~
I I 9 G E P G Q T G P A G A R G P P G P P G K A G E D G H P G K P I 4
A

Bovine

445 g g a g a t g q a g a a g g g t t g t t g 9 a a a g g t t c t g t t t C t g g a a t g g a c t t g g t a a g g a t t a g

Bovine

30

Human

Buaan

61

Bovine

91 aaagcccaatgagataaggaccacgcgaatccaccagccccacaagatgaacgcatcccac

Bovine

Bovine

-1
90

444

534

1 4 9 G R P G E R G V ~ G P Q G A R G F P G T P G L P G F K G I R 1 7 8
Buman
Bovine

535 t c a c a a 9 t c t a t a t t a a c a g c c t g c c c a t t a a t a t t c c t t a a a a c a a c t c c
1

a
G

624

Buman
Bovine

625 c a a a c g a c c c g t g g c t c c g t a g a g a g a c g t g t g t c c c c t c c c a g c g t c c c g t g a a t a t g a a t t
2 0 9 G Q ~ G A R G L P G E R G R V G A P G P A G A R G S

714
D

Human
Bovine

715

ggt~tgtggg~t~tggt~attgg~t~tg~tgg~c~t~cagg~tt~aggtg~t~tgg~aag9gtgaA~t~gga~t~tt

804

2 3 9 G P V G P A G P I G S A G P P G F P G A P G P K G E L G P V 2 6 8
N

Human

Bovine

805 g t a c c t g g t g t g g t g g g g t g t g g g a a g t g g t a g g t t t t g g t g t g a t c t g g a a a

894

Human

269

298

BOVlOl

895 g g a g c c a a t g g g c t t ~ c t ~ t a a 9 g g ~ g ~ t g ~ t ~ g ~ t t ~ g g ~ g ~ g ~ t g g g g c t c ~ g c ~ t ~ t g g a ~ g g g ~ t a ~ t
2

G N ~ G

HULLO
Bovinl

1075

Buman
1165
3

Bulan
Bovine

1255
419

1345
4

1435
4

1525
509

Human
Bovine

Bulaa
Bovine
Buman

Bovi0e

E V G L

G L

V G

T
T

1074
8

ggcgag~tggtg~tgttggg~ag~aggtcctcctggc~cagtggtgaagaaggaaagaqagg~t~a~tggagaaat~a~t
9 G ~ P G A V G Q P G P P G P S G ~ E G K R G B T G ~ I G
$ A
P Q
P N

3
A

1164

T
S

S
A

1344

448

G
L

gg~cagcggga~caa~a9~aga~c~tggcaa~attggatt~cct~ac~aaa~g~c~a~tggtgatc~tgg~aag~tggt~aaaa
9

P A
V

G K
T

a 1524

8
N

~gtcat~ctggt~t~gctg~tgctc~gg~g~t~ag~tcccgat9gcaacaacggt~ctca~gga~t9ga~taca~ggt9tc~a
G
H A
G L
k
G A
R O k
P G
P D G N N
G A
Q G
P
P G
L Q
p

a 1614
538

1704
P

1705 ~ g a g a ~ a g ~ g g t a t c c c t g g t g a ~ t t t g ~ t c ~ c c c t ~ c c c t g c t g g t g ~ a a g a g g g g a g c g g g ~ g c ~ c c c a g g t ~ a a a g t ~ g t ~ c t g c t
5 6 9 G E R G Z P G ~ F G L P G P A G ~ R G E R G p P G ~ S G A ~
L H
P

1794
8

1795 g g g c c t a c t g ~ g c c t a t t ~ g a a ~ c c g a g g ~ c c t t c ~ g ~ a c c c c c ~ g g c c t ~ a t ~ a a a c a a 9 g ~ t 9 ~ c c ~ t g t g g ~ t ~ 9 c ~ t c c
5 9 9 G P T G ~ I G S R G P B G P P G P D G N K G E ~ G V V G A

K
V

a 1884
p

8
V

cactctctccacaccccaaagagtgcggcgcatcagcaagagaaaataaac

1885
2

t
G

19~4

8
p

gg~ctcaga~gtgac~tt9gt~9c~ct~gtagagat~tgct~9t~t9~tcct~gt9ctatt~t~ctcct~t~ctg~ag~aa t

1975
6

Human

Human

ggtt~c~a~ttcc~ctg9aaatatcgg~cc~9ctggtaaagaaggt~tgtgggt~t~t~gtattgacgg~a~ac~tg~g~c~att
1434

Human

Bovine

8
S

ggta9ccgt~tgcaa~tgg~ctgctggtgtg~gagg~c~atgga~a~t~tggt~g~cct~a~agcctg~c~t~at~a~cc~a

1615 g ~ t g ~ a ~ g g t g a a c ~ g ~ t ~ t ~ c t g g t ~ t c c a g ~ c t t c c a g ~ g t ~ t g ~ c t g g c ~ c t ~ c a ~ c a c a g c t g ~ t g a a g c t ~ g ~ a a a c c a
5 3 9 G G K G ~ Q G P A ~ P P O F Q O L P G P A O T A G E ~
S
P

Bovloe

g ~ a g g ~ t ~ c t g g g ~ t g a g g g g a a a t c c t g g c t c c ~ g t g ~ t ~ t a c c t 9 ~ a g c t g a ~ g g ~ a g a g c t g g t g t c a t g g g t ~ c t1254
~t
9 G P P G P P G L R G N P G S R G L P G A D G R A G V M G P A 4 1 8
S
p

Human
Bovine

G N ~

984
I

Bulan
Bovine

Human
Bovine

A G

Human
Bovine

P G
T

Bovine

985 g g ~ t g t ~ g g ~ g ~ t g ~ g t g ~ a ~ g g c g ~ c ~ g ~ g g a c t t g t t ~ g t g a g ~ g g ~ c ~ a g ~ t g g t t c g a a a g g a ~ a 9 a ~ c g g c a a c a a ~

Human
Bovinl

~ G

D
E

P
N

G
H

G P
V

2064

8
T

~ggga~cg~gt~aag~tgg~cccgct~9ccctgct~gccc~9ctggt~tcgtg~ta~t~t~aac~tg~t~a9gtc~tccc~ct 2154
6 B g G D R G E A G P A G P A G P A G P R G S P G E R G E V G P A T I 8

2065

Bovine Proct2(I) c D N A
Bovine

2155 ccccaacgatttcttcctgctgtctctgtcacctgtctaaggggaggaaccaagacccaagggt
7 1 9 G P N G F A G P ~ Q ~ A G Q P G ~ K G ~ R G T K G

87

emaat
7 4

2244
8

Human
Bovine

2245

ggkcct~ttggtcccacaggccccgttgga~c~gccggtccgtct~tccaaat~gcccacctggtcct9~tggaagtcgtggtgatgga

2335
7 7

ggg~tgg~gct~t~t~t~cct~gt~tgctg~c~ga~tggtcccc~t9~ac~tctggt~tct~t~c~t~gcc~ccct

Human
Bovine

Human
Bovine

2424
0

2514

2604
0

~
2605 ggttttctgtctccctctaagtacttctaccaggt9tcgctggatctgtgggtaacctggccccccgcatcgca
6

Bum an

2694

X
2695 ~g~cc~cc~ccc~t~gtccccct~gt~tgtc~cctg9cgtc~at~9t~ctcctggtg~a~ccggtc~tg~cggc~ccct
8
9
9
~
p
p
~
p
p
~
V
~
N
~
V
N
G
~
P
~
E
A
O

H um an
Bovine

2515 g~ctttgtt~tg~g~tccctct~a~gcctg~ac~ctgg~cctc~t~aa~cccaggtccacaaggccttcttggtgctcct
8 3 9 G F V G E K G p S G E p G T k G P P G T P G P Q

Bovine

Human
Bovine

2425 ~gtcctgct~gt~aaga~gggcttcgt9g~cctc~t~gtga~c~ggtc~agt~ggtcga~gtg~agagac~ggtgcctctg~cc~tcct

Human
Bovine

2334

2794
~

~
2705 gg~a~cggtcccccag~cgc~tggtcaacccggacacaag~ggg~gcgtggttaccccggta~c~c~gt~ct~ttggtgctgcc
838 ~ N D m ~
P P ~
)Pt D ~ Q P ( ~ ]HI 14~ 40 ~ ]PL ( ~ Y ~ O LNI ~
~;

~,r ( 3

2874
958

2964
980

101B

3144
1048

3234
1078

Human
Bovine

2875
959

tctcctgccctcagctttcccgttgtaaacgaaaccgtggtaccgtctgcctctgttgtcctgct
~

Human
Hovine

Ip. Q
H

~r

~r
~

I~

G ~ V G

R G

Human

Human
Bovine
Human
Bovine
Human

Figure 1. Nucleotide sequence of

the bovine proc~2(I) cDNA and
its deduced amino acid sequence.
The nucleotides are numbered
from the translation start site.
The bottom line is the amino
acid
sequence
for
human
pro0~2(I) where it differs from
the bovine sequence. Amino acid
residues which are different from
the published ones in the SWISSPROT database with accession
number P02465 are underlined.
The regions of the deduced
amino acid sequence that correspond to residues 308-319,
509-523, 620-629, 746-765,
944-958 and 1013-1020 were
confirmed by microsequencing
of lysylendoproteolytic peptides
from purified bovine 0~2(I) chain
(unpublished data). Dashes indicate gaps inserted to achieve optimal alignment. Asterisk indicates stop codon for translation.

Bovine

I~

I~

~
T

~,
~

4~ A
P

D
E

3054

3055 ~ctt~aaggg~c~caat~ggttgcaa~tctcccgggtcttgctggtc~tcatggc~tc~gt~ctcccggtgctgtgggtccc~ct
1019 O L K G H N O L
Q G L
P G L
Jk O H H G D Q G ~
P ~ A V G

Human
Bovine

2965 ggtgccttggcccaagagtcccagtggcccacaggtattcgggtgaaagggagcctggtgtaaggtcccaaggtcttcct
989

Bovine

P"

3145 ggtccc~gg9gccct9ct~gtccttctggcccc~ctggc~a~gacggtcgcattggacagcctggtgc~gtcggacctgctggc~ttcgt
1045 ~
p R ~
P 2~ ~
P 8 ~
P 2~ 4~ K b ~ 1~ X ~ Q P G ~
~ ~
P
T
H
T

2~ ~

3235 ~gctctc~g~gtagc~aaggt~ctgctggccctcctg~tccccct~g~c~tcct~gacctcctggcccaagtggtggtg~ttacgagttt
1 0 7 8 G 8 Q G $ Q (~ p A ( ~
P P G P P ~
P P ~
P P (~ P 8 G O O
P
H
V

E
D

3308

3325 ~gttttga~ggag~cttct~cagggc~g~cc~gcctcgctcaccaacttctctcagaccca~ggatt~t9~gttgatgct~ctctgaa~
1109 G F D G D F Y R 2 ~ D
Q P R S P T 8 L R P K D Y E V D2q.
Y
.~ P

3414
1138

3 6 1 5 tctctcacacgttggcccttcttactccgaaggctctgagaacccagcc9cactgcc9agactgagactc9ccc
1 1 3 9 S L M M Q I ~ T L L T P E O S R K N P A R T C R D L R

3324

3504

O
P

3594
8

E
L

3684
8

Human
Bovine
Suman

3505 ccagaatgggcagtggttactactggattaccctaccaaggattctatggatgtatcaaagtatactgtgatttctctactggc
3 1 6 9 p ~ H B G y y W I D p N Q G C T M D A X K V Y C D W
E

Bovine

2595
l

Human
Bovine
Human
Bovine

Human
Bovine

gacctcatccgctcaacctaaactcccagtcagaactggtacaaattccaggccgctgtctg9tagga9aa
9 9 E T C Z ~ Q P E D Z P V K N W Y ~ N S K A K K ~ V ~
N
~
S
D

3685 ~ct~tca~cgg~ggt~ccc~gtttgaatat~tgttga~ggagt~accaccaaggaaatggct~c~caa~ttgcctt~atgcgt~tg~tg
1 2 2 8 T I N G G T Q F Z Y N V E G V T T K E M k T Q L A F M R
~
S
S

3775 gccaaccatgcctctcag~tcacctacc~ttg~a~c~gc~ttgcatacat~gatg~gg~aactggcaacctgaaaaa9gtgtc
| 2 5 9 R N H ~ B Q N I T Y H C K H S X ~ Y M D E E T G N L K K ~
y

3865 ttctgcaagatcctgatgtcacttgttgccgagggcaccagttcacttcactgttcttgtagtggctgctctaaag
1 2 8 9 I ~ G N N D V E L V A E G N H R F T Y T V L V D G

3774
8

3844
V

3954
8

4044
8

Human
Bovine
Human

3955 acaa~tg~atggc~gacaatcattgaat~taaaacaaacaagccatctcgcctgcct~tccttgatattgc~cctttgg~c~tcggt
1 3 1 9 T N E N Q K T X l E Y K T N K P S R L P I L D I k P L
O
T

Bovine

4045 ggcgctg~cc~g~tc~gattgaacattggc~cagtctgtttcaaat~a~c~a~ctca~cctaa~ttaaagaaa~agg~tctga~

4134

F~VD

Human
Bovine

4135
4225
4315
4405
4495

catttct~ttggcc~tt~ctttttcttctttcctaa~tg~aa~ctgaatccttcc~tttcttct~cacatct~cttgctt~ttgtggc
aa~aga~gag~gg~ttg~t~a~agcattgtgc~tacaattta~ttcactccccctcccttttcccctctc~a~a~gatttgg~tttt
~ttttttt~cactctt~c~cctgttgt9g~aaat~t~aacctttgtaa~aa~c~t~a~a~t~aa~ataa~aac~tga~c~
tttgc~cc~ctt~tggcttttg~atatcttccac~ggg~agtttaaaacccaaacttccaaaggtttaaact~cctc~a~ca~tttc
ctgtgagtg~gatccecacctcgt

4224
4314
4404
4494
4518

88

T. Shirai et al.

and the complete eDNA sequence was determined by

the dideoxy-chain-termination method using Sequenase
V.2.0 (U.S.Biochemical Corp.) and BcaBEST (TaKaRa).
A full-length cDNA coding sequence of bovine
pro0~2(I) and its deduced amino acid sequence are
shown in Figure 12, together with the published human
proc~2(I) sequence for comparison (de Wet et al., 1987).
The bovine proc~2(I) chain is 1364 amino acids, and its
amino acid sequence is highly homologous to the human
and mouse sequences (Phillips et al., 1992), with identities of 92% and 89%, respectively. Another characteristic feature is that methionine at residue 783 in the
human and mouse pro0~2(l) chain is replaced with alanine in the bovine chain.
The existence of polymorphic pro0~2(I) mRNA transcripts was demonstrated in both human and chicken fibroblasts (de Wet et a1.,1987). Filter-bound RNAs from
bovine and human cultured fibroblasts were hybridized
with [0t-~eP]-Iabelled probe specific for the correspond*
ing proc~2(I) cDNA (Bernard et al., 1983). Northern blot
analysis showed multiple bands for bovine pro(x2(I)
mRNA as in the case of human mRNA. Among them,
5.6 kb and 7.0 kb transcripts showed the same mobility
as those of human mRNA transcripts (data not shown).

References
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Dr. Hisae Hnri, Division of Adult Diseases, Medical Research
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Received November 11, 1997; accepted February 5, 1998