Download Proposed alignment of helical interruptions in the two subunits of the

September 1986
FEBS 4028
Volume 206, number 1
Proposed alignment of helical interruptions in the two
subunits of the basement membrane (type IV) collagen
Gabriel
Vogeli, Elizabeth
Horn+, Jane Carter*
and Paul S. Kaytes
Molecular Biology Research and *Biopolymer Chemistry Research, The Upjohn Company, Kalamazoo, MI49001,
USA
Received 1 August 1986
We have isolated a cDNA clone for part of the a2 type IV collagen (pCIV-Z 176). Deoxynucleotide sequence
analysis shows that this clone codes for 439 amino acids from the helical domain adjacent to the C-terminal
globular domain of the a2 (IV) chain. By aligning the deduced amino acid sequence of the a2 (IV) chain
with the published sequence for the al (IV) chain, we find that all interruptions in the aI (IV) chain coincide
with an interruption in the a2 (IV) chain. Additonal interruptions in the a2 (IV) chain exist, however, three
out of the four analysed only slightly disturb the collagen triple helix.
Mouse
Collagen
DNA sequencing
Helical interruptions
1. INTRODUCTION
Basement membranes are essential histological
and physiological barriers in animals and humans
[1,2]. Type IV collagen that forms the scaffold of
the basement membrane [3] has two cy1 (IV) and
one 1x2(IV) subunit [4]. The central part of each
subunit consists of a Gly-X-Y repeat, that unlike
the interstitial collagens, is interrupted in the LYI
(IV) collagen chain (51. To determine whether such
helical interruptions also exist in the ~2 (IV) collagen chain, and to analyse their position relative
to the a1 (IV) chain [5-81, we characterized a partial cDNA clone for the a2 (IV) chain.
2. MATERIALS
AND METHODS
The cDNA clone pCIV-2-176 was isolated from
a mouse Engelbreth-Holm-Swarm
(EHS) tumor
cDNA library (in the PstI site of pBR322) [7] by
hybridizing the cDNA library [9] at low stringency
+ Present
address:
20879, USA
Oncor,
Inc.,
Gaithersburg,
MD
Basement membrane
Alignment
(e.g. 0.9 M NaCl, 30% formamide at 42°C for
16 h, washed in 15 mM NaCl, 1.5 mM Na citrate
at 25°C) with pCIV-1-225, an (~1(IV) cDNA clone
[7]. The hybridization signal from pCIV-2-176 was
removed at high stringency (washing at 51 “C).
Standard protocols [lo] were used to prepare,
subclone and sequence the DNA.
3. RESULTS AND DISCUSSION
pCIV-2-176 was isolated from a cDNA library
[7] prepared from RNA isolated from the
Engelbreth-Holm-Swarm
(EHS) tumor, tissue that
is rich in basement membrane components [l 11.
We hybridized the cDNA library under nonrestrictive conditions with labelled DNA from
pCIV-1-225, an (~1 (IV) cDNA clone [7]. We
argued that under these conditions cDNA clones
related to collagen will hybridize, and that some of
the positive colonies will code for the cy2(IV) collagen chain. The identity of pCIV-2-176 as an cy2
(IV) collagen clone was established by sequence
comparison (Kuhn, K. and Timpl, R., personal
communication). pCIV-2- 176 also overlaps at its
Published by Elsevier Science Publishers B. V. (Biomedical Division)
00145793/86/$3.50 0 1986 Federation of European Biochemical Societies
29
Volume 206, number 1
DNA
GGcGcK;Gcm
a2
.
al
l-lmxmxmm
L
P
.
F
P
~~l-mx%xA~mmm
.
v
S
.kgP
I T .
.
120
F
P
.
. Asp
F
. ASP
1 .
Asp
X
.
X
. GJu
X
CM
G~X;GTGACATTGGR~CC~;KZ~~ZT~
A
-hP
A
T
P
.
V
A
.
V
F
.GJuT
.
L
P
.
S
P
.
L Lys
. GluArg
.
T
.
I
P
.
L ~ys
.
F
F
. GduLys
.
.
P
P
.
I .
I
P
.
L kg
. G3u
. AspGJn
.
I
A
.
F
.
S
p
. Gju
. GJu
.
AGln.
S
P
.
LLys.GlnT
.
T
.
LGln.
P
T
.
aa
X
I
243
v ASP
P
X
A
1
A
al
X.Sl.lP.MP.SP.LX.SP.SV.YP.SP.LP.Gd~X.~pLys.LP.L
*ASP’
.
F
P
.
1
T
.
M
A
AAmx&KNxcll-mK
369
F
P
.
L
. Glu
P
.
S Asp
.
I
P
.
PGln_GduP
,kg
S
. GluLys
. Glu
ATCEA4TMXO3Zr~TG8
a2
Asp .
I I’ .
V X . Glu A .
L f’ .
T P
.
P
A
615
ccmxmGATaxcA~~
ASP P
al
. GlnLys
.
03
F
P
.
F
P
.
P
T
. AspArg
.
F
P
.
S Lys . AspLys
.
LGln.
621
. Aspkg
. Glu
A hsn
T
L
P
.
P
V
.
V
P
. GluLys
. Glukg
.
.
. Glu
V
-
F
P
.
L
A
.
S
P
.
.
. Glu
S Lys
~~K~~;GM~;~~I~~~L~~~~~~TA~Tw+Iu~TGT~TA~K
P
.GluArg.
Gin
.
A
(B)
TGKXXXXGK;F
a2
a2
at
Skg.GluLys.
T
d”
. Gtn
a2
DNA
.
l-E-T
T
DFH
T
~~A-MTATT~~;-~~~~~
p
al
F
zm 469
a2
a1
DNA
September 1986
.AP.LP.PP.SV.GM.LP.TP.G#~X.VP.IP.PGJ~.SP.LP.
(a)
cnv!
FEBS LETTERS
P
A
.
S
.
P
P
.
P Gln
. Gln
F
.
I Am.
F
M
P
P
.
P
P
P
M
.
P
P
.
P
P
.
F
P
.,+,.SP.kpV.AP.lF.LG’..Y
L
P
.
T
P
.
-His
P
.
0
I
P
v LYS
T
747
V Glu
.
P Lys
. kspkg
W
P
.
A
P
.
S
.
A
V
. Aspks
.
P Gln
. Gln
P
.
L
P
.
FGln.
. His
.
PLys.AspP
.
P Lys
.
P Lys
. AspGIn
.
.
L Gln
.
V Lys
. AspGIn
. Asp
L
P
.
A
L
P
P
.
WA
a2
Gln.
al
P
ONA
.
P
PLys.AspLys.AsnGln.
CCM%TKKQ3
.
V
L
.
L
P
. GIuLys
.
P Lys
. GluGln
.
F
H
. Asn
T
.
P
al
P
.
I
G
.
S
P
.
.
S Lys
. Asp
.
L
P
.
P
.
F Gln
P
.AP.SM.SP.IP
I
T
H
V
. GlnLys
.
L
C
P
F
F
al
Gln .
DNA
aX;CK
a2
PGln.
a1
P
P
v
.
P
.
P Lys.
LGln
P
P
.AspP
.
FN-g.
. GInGIn
.
V
P Lys
.
T
P
.
P
.
P
P/rg
A
P
.LysA
S
V
.
L
P
Y AspV
.
PGln
.
P
I Lys
07
P
. GluP
P
al
Pkg.
v
. GInGlu.
F
P
GluP
.
P
. Arg
P
.
Pkp.
P
.
.
L
P
.
PGlu
.
.GluI
.
L LYS
.
.
P
M
.HisGln.
P
F
.
~k-g
1251
.N-gG
P
.
S
.mi.
P
.
FArg.kpGln.
F Asp
.
08
A
P
.
GlnLys
s
I
g
y
I
I
vlys
g
f
I
v
~~TG===g~+gtgw=tcggct=t~tggts=g
a2
I 129
.1--g.
1317
DNA
M
999
P
DNA
P
cl-rc
a2
a2
V
S
P
.
L
P
.
H
P
.argsv
L
P
.
S
H
.
P
P
.
t
p
s
v asphis
. Glu
T
.
P
P
.
1347
t
(0
Fig. 1. Deoxynucleotide sequence of pCIV-2-176, a cDNA clone for the LYZ
(IV) collagen chain. The DNA sequence for
the cy2(IV) collagen chain is shown in the first lane (the numbering starts arbitrarily at the 5’-end of the clone), the
derived amino acid sequence in the second lane. Line 3 shows the amino acid sequence for the cyr (IV) collagen chain;
from position A to B part of from the human CYI(IV) sequence (amino acid no.469 to 615 (81); followed by data from
the mouse [7] to C and to the end [6]. The glycines essential for the typical collagen Gly-X-Y repeat are represented
by a dot; the charged amino acids are written in the three-letter code; small letters indicate the C-terminal globular
domain. The interruptions of the Gly-X-Y repeat in the cy2(IV) chain are numbered. ‘Missing’ amino acids invoked
to bring the Gly-X-Y repeat in phase after an interruption are marked by a dash.
30
Volume 206, number 1
FEBS LETTERS
N-terminal end with a published a2 (IV) cDNA
clone [ 121.
The DNA sequence from pCIV-2-176 for 1317
nucleotides and the deduced amino acid sequence
for 439 amino acids of the helical Gly-X-Y repeat
adjacent to the C-terminal globular domain are
shown in fig. 1. The border between the helical portion and the C-terminal domain is considered the
nucleation point for triple helix formation in collagen [2]. Since pCIV-2-176 contains this area we
tested the alignment of the amino acid sequence of
the LYZ
(IV) subunit with the published sequence of
the crl (IV) subunit [6-81 starting with the last
glycine in the last Gly-X-Y repeat of both subunits
(see fig. 1).
This portion of the (~2 (IV) collagen chain contains 8 interruptions, the LYI(IV) chain only four.
All interruptions seen in the (~1 (IV) chain (nos
1,3,5,7) that lack complete Gly-X-Y repeat units
are also present at the same relative position in the
a~ (IV) chain. Three interruptions only found in
the (~2 (IV) chain consist of a simple substitution
for glycine in the Gly-X-Y repeat (nos 2,6,8), and
one interruption (no.4) has an additional amino
acid.
We find that our alignment of the two subunits
positions the interruptions in the cri (IV) collagen
chain opposite interruptions in the LYE
(IV) collagen
chain. Our alignment is based upon (i) beginning
the alignment at the nucleation point, and (ii) maximizing the length of the Gly-X-Y repeats between
the interruptions. In addition, by using the human
cri (IV) collagen sequence (fig.1 from the Nterminus to point B), we find that interruption
no.1 in the cyz (IV) of the mouse coincides with an
interruption in ai (IV) from human. Our alignment of the two subunits is further supported by
the fact that one interruption in the LYZ(IV) chain
73 amino acids further to the 5 ‘-end, contained in
a overlapping cDNA clone [ 121, is opposite an interruption in the LYI(IV) chain [8] (not shown). For
the above reasons, we assume that the register we
have chosen for the presentation of our data (fig. 1)
is the most probable alignment.
The flexibility of the type IV collagen molecule
seen in the electron microscope [ 131 can be explained by our finding that helical interruptions
are opposite each other. Since the major interruptions (1,3,5,7) in the Gly-X-Y repeats of the two
different chains of type IV collagen are in identical
September 1986
positions, we propose that the genes for the two
collagen chains arose from the duplication of an
ancestral gene which contained the common interruptions (1,3,5,7). We could then assume that the
ancestral cyz (IV) chain underwent further mutations in the course of evolution. We find that single
base pair substitutions can account for the appearance of three (nos 2,4,8) of the 4 unique interruptions in the a2 (IV) chain. In interruption no.2,
Ser is substituted for Gly and in interruption nos 6
and 8, Ala is substituted for Gly. Either one of
these substitutions will not affect the physical
parameters of the collagen molecule substantially
(Mao, B. and Vogeli, G., unpublished data from
computer modelling). It is, however, also possible
that the cyz (IV) chain was the ancestral gene with
many interruptions that were then repaired to form
an cyi (IV) subunit able to form a more stable triple
helix. Further support for a common ancestral
gene comes from the finding that some of the
regions between the interruptions have a high
homology to each other and that the C-terminal
end of both chains has a high homology (Kaytes,
P.S., unpublished).
During the submission of this manuscript a
report of clones similar to ours appeared in the
literature: Schwarz et al. (1986) Eur. J. Biochem.
157, 49-56.
ACKNOWLEDGEMENTS
We thank Dr R. Timpl and Dr K. Kuhn for
amino acid sequencing data of the cyz(IV) collagen;
Boryeu Mao for advice on the 3 dimensional structure of collagen and Kathy Hiestand for help with
the manuscript. The present work had been initiated by G.V. and E.H. at the Laboratory for
Developmental and Molecular Biology, National
Eye Institute, Bethesda, MD 20205.
REFERENCES
111Laurie, G.W. and Leblond, C.P. (1983) J.
Histochem. Cytochem. 31, 159-163.
121Pi% K.A. and Reddi, A.H. (1984) Extracellular
Matrix Biochemistry, Elsevier, Amsterdam, New
York.
131 Tim&
R., Wiedemann, H., Van Delden, v.,
Furthmayr, H. and Kuhn, K. (1981) Eur. J. Biothem. 120, 203-211.
31
Volume 206, number 1
FEBS LETTERS
[4] Tryggvason, K., Robey, P.G. and Martin, G.
(1980) Biochemistry 19, 1284-1289.
[5] Schuppan, D., Timpl, R. and Glanville, R.W.
(1980) FEBS Lett. 115, 297-299.
[6] Oberbaumer,
I., Laurent,
M., Schwarz, U.,
Sakurai, Y., Yamada, Y., Vogeli, G., Voss, X.,
Siebold, B., Glanville, R.W. and Kuhn, K. (1985)
Eur. J. Biochem. 147, 217-224.
[7] Nath, P., Laurent, M., Horn, E., Sobel, M.E.,
Zon, G. and Vogeli, G. (1986) Gene, in press.
[8] Babel, W. and Glanville, R.W. (1984) Eur. J. Biothem. 143, 545-556.
32
September 1986
[9] Vogeli, G., Horn, E., Laurent, M. and Nath, P.
(1985) Anal. Biochem. 151, 442-444.
[lo] Maniatis, T., Frish, E.F. and Sambrook, J. (1982)
Cold Spring Harbor Laboratory, NY.
[ll] Orkin, R.W., Gehron, P., McGoodwin, E.B.,
Martin, G.R., Valentine, T. and Swarm, R. (1977)
J. Exp. Med. 145, 204-220.
[12] Kurkinen, M., Bernard, M.P., Barlow, D.P. and
Chow, L.T. (1985) Nature 317, 177-179.
[ 131 Hofmann, H., Voss, T. and Kuhn, K. (1984) J.
Mol. Biol. 172, 325-343.