Vol. 263, No. 32, Issue of November 15,pp. 17159-17166,1988
Printed in U.S.A.

THEJOURNAL
OF BIOLOGICALCHEMISTRY

0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Cloning andSequencing of Pro-al(X1) Collagen cDNADemonstrates
That Type XI Belongs to the Fibrillar Class of Collagens andReveals
That the Expression of the Gene Is Not Restricted to Cartilagenous
Tissue*(Received for publication, February 26, 1988)

Michael Bernard$, Hidekatsu YoshiokaS, Elida Rodriguez& Michel van der RestQll,
Tomoatsu Kimurall, Yoshifumi Ninomiyall,Bjorn R. OlsenII,and Francesco Ramirez$
From the $Department of Microbiology and Immunology, Morse Institute of Molecular Genetics, State Universityof New York,
Health Science Center at Brooklyn, Brooklyn, New York, 11203, the §Genetics Unit, Shriners Hospital, Montreal,Quebec,
Canada H3GlA6, and the IlDepartment of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115

We have isolated several overlapping cDNA clones that the threechains are assembled into one parental tripleencoding al(X1) collagen chains from human and rat helical molecule, called Type XI collagen (Morris and BachcDNA libraries. Together the human cDNAs code for inger, 1987). The l a , 2a, and 3a chains have therefore been
335 uninterrupted Gly-X-Ytriplets, and a 264-amino re-named al(XI), &(XI),and a3(XI), respectively (Eyre andacid C-propeptide,while the rat cDNAs cover theen- Wu, 1987). The genetic identity of the a3(XI) chain, however,tire C-propeptide and about a third of
triple-helicaltheremains controversial, because on peptide mapping this chain
domain. Comparisonof the human and rodent nucleo- exhibits a pattern similar, if not identical, to that of the a1
tide sequences showed 95%
asequence similarity. The chain of Type I1 collagen (Burgeson et al., 1982). The al(X1)identification of the clones
as crl(X1) cDNAs was based and a2(XI) chains, on the otherhand,aredistinct
gene
on the complete identity between the amino acidse- products which are closely related to the al(V) and a2(V)quences of three humanal(X1) cyanogen bromide
pep- chains, respectively (Burgeson et al., 1982; Eyre and Wu,
tides and the cDNA-derived sequence. Examination of1987).
the cDNA-derived amino acid sequence showed a vaFunctionally, Type XI collagen has been implicated inriety of structural features characteristic of fibrillarregulation of the diameter of Type I1 collagen fibrils and informing collagens. In addition,nucleotidesequencecol1agen:proteoglycaninteractions (Sheren et al., 1986; Smith
analysis of aselectedportionofthecorresponding
etal., 1985). Theseandotherpossible functions may behuman gene revealed the characteristic 54-base pairexon motif. We conclude therefore thatpro-al(X1)col- limited to thepericellular region of chondrocytes, albeit suchlagen belongs to the group of fibrillar collagen genes.a conclusion rests on immunological studies possibly hamWe also suggest that the expressionof this gene is not pered by the limited epitope accessibility in the matrix, as
restricted to cartilage, as previously thought,since the observed for Type V collagen (Fitch et al., 1984; Ricard-Blum
cDNA libraries from which the clones were isolated, et al., 1982).
The concomitant expression of Types I1 and XI in all
originated from both cartilagenous and noncartilagicartilage tissues closely resembles that of Types I and V innous tissues.
non-cartilage matrices (Eyre andWu, 1987). However, unlike
Type XI, which has never been detected in a tissue lacking
Type 11, Type V collagen is not exclusively associated with
Burgeson and Hollister (1979) first reported the identifi- Type I. There are infact, at least two cases in which Type V
cation of a minor collagen component in cartilage which upon collagen is found associated with Type I1 rather than Type I
SDS-PAGE’ fractionation yielded three distinct chains, pro- (Eyre and Wu, 1987). Types V and XI are therefore believed
visionally named la, 2a, and 3a. More recent data suggest to be closely related collagens not only structurally but alsofunctionally, in that both are minor matrix components ex* This work wassupported by National Institutes of Health Grants hibiting functions that are ancillary to those of the major
AR-38648, R01 HD 22657, AR 36819,and AR 36820, March of Dimes, tissue collagens, Types I and I1 (Eyre and Wu, 1987).
The isolation of pro-al(X1) clones from two mammalian
Birth Defects Foundation Grant 1-1042, Canadian MRC Grant MT7796, and the Shrinersof North America. The costs of publication of species has now enabled us to obtain novel and important
this article were defrayed in part by the payment of page charges. information regarding the structure, evolution, and expression
This article must therefore be hereby marked “advertisement” in of Type XI collagen. In addition to providing the amino acidaccordance with 18 U.S.C. Section 1734 solelyto indicate this fact.
The nucleotide sequence(s) reported in thispaperhas been submitted sequence of the C-propeptide and most of the human al(X1)totheGenBankTM/EMBLDataBankwith
accession number(s) chain, our studies confirm the fibrillar nature of Type XI
collagen and suggest that the expression of COLllAl is not
504177.
11 Senior scholar from the Fonds de la Recherche en Sa& du restricted to cartilage. Finally, the availability of Type XIQuebec.
probes should help ascertaining the potential role of thisThe abbreviations used are: SDS-PAGE, sodium dodecyl sulfate- collagen in human cartilage disorders, such as chondrodystropolyacrylamide gel electrophoresis; bp, base pairs; C-propeptide, carboxyl-terminal propeptide; C-protease, the enzyme that specifically phies and osteoarthritis.
cleaves the carboxyl-terminal propeptide; COLlAl, COLlA2,
MATERIALS ANDMETHODSCOL2A1, COL3A1, COL5A2
and COLllAl, pro-al(I),pro-a2(1), proal(II), pro-al(III), pro-a2(V), and pro-al(X1)collagen genes, respeccDNA and Genomic Clones-Three human and one rodent cDNA
tively; HPLC, high performance liquid chromatography.
libraries were used in these studies. One of the human libraries (a17159

17160Mammalian Pro-al(XI)(COLI
CollagenGene

generous gift of Drs. P. Berg and H. Okayama, Stanford University)
was generated from RNA purified from an SV40-transformed fibroblast cell line (GM 637) (Okayama, and Berg, 1983). The second wasconstructed in Xgtll, using as template poly(A+) RNA from shortterm cultured costal chondrocytes as described? The third was a
Xgtll placental tissue cDNA library purchased from Clontech (Palo
Alto, CA). The RNA from a Swarm rat chondrosarcoma was thesource of the rodent cDNA library which was constructed in the Xgtll
vector as described?
The positive-negative screening protocol which led to theidentification of the first Type XI clone, OK4, from the GM 637 cDNA
library has been detailed elsewhere (Weil et al., 1987). It should be
noted that, lacking conclusive evidence, in that report we referred to
OK4 as a Type V-related cDNA. The screenings of the cDNA libraries, as well as theprocessing and analysis of the positive clones were
essentially as described (Weil et al., 1987).'
The genomic library was constructed in the Charon 4A vector andit contained human DNA partially digested with EcoRI (Ramirez etal., 1979). Screening, isolation, and purification of the phage clones
were performed as described previously (Chu et al., 1984).
Nucleotide Sequence Analysis-DNA sequencing was carried out
with the dideoxy chain termination technique (Sanger et al., 1977)
eitheron single stranded DNA in M13 vectors using a standard
protocol (Vieira and Messing, 1982), or on double stranded DNA in
pUC vectors following modifications of the procedures described by
Hattori and Sakaki (1986) and Zagursky et al. (1986). Sequencing ofboth strands was performed for the majority of the sequences presented here.
Collagen Purification-Cartilage collagens were prepared by a modification of the method of Burgeson and Hollister (1979). Human
articular cartilages were obtained at autopsy from infants (<4 yearsof age) who died from disorders unrelated to connective tissues. The
insoluble residues, left after a 4M guanidine hydrochloride extractionof the proteoglycans (Roughley and White, 1980), were powderizeda t liquid nitrogen temperature in a freezer-mill (SPEX Industries,
Metuchen, NJ). Thepowder was resuspended (300 mg/ml) in cold 4
M guanidine hydrochloride, 50 mM Tris-HC1, pH 7.4, and extracted
for 48 h at 4 "C under constant stirring. The insoluble residue wascollected by centrifugation (12,000 X g, 10 min), washed four times
with cold distilled water, and resuspended (150 mg/ml) in a cold 0.5
M acetic acid, 0.2 M NaCl solution, and treated with pepsin (1 mg/ml) for 72 h. The pH was then raised to 8.1 with NaOH to denature
pepsin. The viscous solution was clarified by centrifugation (24,000
X g, 30 rnin), and the supernatantwas dialyzed for 72 h against threechanges of a 0.5 M acetic acid, 0.9 M NaCl solution. The precipitated
Type I1 collagen was pelleted by centrifugation (24,000 X g, 30 min).
The supernatant was filtered through Whatman No. 1 paper anddialyzed for 72 h against three changes of a 0.5 M acetic acid, 1.2 M
NaCl solution. The precipitated Type XI collagen was collected bycentrifugation as above. The same procedure was further applied toprecipitate the Type IX collagen at 2.0 M NaCl in 0.5 M acetic acid.
The purified collagens were dialyzed against 0.5 M acetic acid andlyophilized.
Separation of the Type XI Chains by CM-cellulose Chromatography-Type XI collagen (8 mg) wasdissolved in 8 ml of 0.04 M sodium
acetate, pH 4.8, buffer containing 6 M urea, denatured by heating at
42 "C for 10 min, and loaded on a 1.6 X 14-cm CM-cellulose (Whatman CM52) column, heated to 4 2 T , and equilibrated with the same
buffer. Elution was performed with a NaCl gradient (0-0.09 M, 500ml) in the same buffer and monitored with a Schoeffel 770 monitor
set at 230 nm.
Gel Electrophoresis-SDS-PAGE was performed in 7 cm X 8 cm X
0.75-mm (length X width X depth) gels in a mini Protean I1 cell(BioRad) according to Laemmli (1970).
High Performance Liquid Chromatography (HPLC)-Separation of
CNBr-derived peptides was done as described previously (van derRest and Fietzek, 1982) on a Vydac TP 201 (4.6 X 250 mm) reversedphase column (The Separation Group). The initial separation was
done using 10 mM heptafluorobutyric acid as ion-pairing agent, andthe peptides were further purified by a second chromatography stepwith 9 mM trifluoroacetic acid. The equipment used was from Beckman and consisted of a Model 334 chromatograph, a Model 160 UV
monitor equipped with a zinc lamp, and a CR-1B data system.
Amino AcidSequence Analysis-Amino acid sequences were determined by automated Edman degradation in an Applied Biosystems'T. Kimura, M. G . Mattei, J. W. Stevens, M.B. Goldring, Y.
Ninomiya, and B. R. Olsen, manuscript submitted for publication.

1Al)

Model 470A sequenator, using the trifluoroacetic acid conversion
program. The phenylthiohydantoin derivatives of the amino acids
were identified by HPLC as described by Lazure et al. (1983).
Cleavage with Cyanogen Bromide-The
purified chains were dissolved in 70% formic acid containing 12 mg/ml CNBr, andthe
solutions were flushed with nitrogen. After a 16-h digestion a t room
temperature, the solution was diluted four times with water andfreeze-dried.
RESULTS

Isolation and Characterization of Type V-related cDNA
Clones-We have already documented the isolation of a distinct collagen-coding recombinant, OK4, from the GM 637
library (Weil et aZ., 1987). The insert of this clone is nearly
4.2 kilobases in length and contains a 579-nucleotide long 3'untranslated region which includes a shortpoly(A) tail, a792bp portion coding for a cysteine-containing globular domain,
and a2812-bp portion coding for an uninterrupted collagenous
sequence (Fig. 1).Based on the similarity between the aminoacid composition of OK4 and that of the a l ( V ) chain, we
provisionally referred to this clone as coding for a Type Vrelated gene product (Weil et al., 1987). In order to obtainfurther 5' sequences, an appropriate subfragment of OK4 wasutilized to screen 5 X lo4 phage plaques of the Xgtll humanplacental tissue cDNA library. This resulted in the identification of two positive phages, of which one, Hpl 1, was found
to extend an additional 202 bp beyond the 5' end of OK4 (Fig.
1).At the same time, and independently of these experiments,
some of us (T. K., Y. N., and B. R. 0.) isolated from humanand rat cartilage-specific libraries several clones whose sequences were found to be identical and homologous to OK4,
respectively. The restriction map of the largest human chondrocyte clone, KTh 98, is shown in Fig. 1, whereas Fig. 2
shows a compositus of the nucleotide sequence and conceptual
amino acid translation of the threeoverlapping human clones.
Fig. 3 shows a comparison between the common coding sequences of the human and the largest rat clone, pKT 1201.
This analysis showed a level of amino acid divergence of only
6% in the C-propeptide and 5% in thetriple helical domain.
Collectively, these data suggested that the Type V-related
gene product encoded by OK4 is expressed in at least four
different tissues, of which two are cartilageneous. This raised
the strong possibility that thecDNAs may codefor one of theType XI collagen chains. Hence, biochemical analyses were
performed to prove the validity of this hypothesis.

'3"5

Klh98

t

t1 Kb

I
pKT 1201I

FIG. 1. Restrictionendonuclease map of the mammalianC O L l l A l cDNA clones. On top is the relative location of the threeprotein domains encoded by the cDNAs, namely C-propeptide (C),telopeptide ( T ) , and achain (A). Underneath is the restriction mapof the human clones, whose relative location is depicted along withthat of the rat cDNA, pKT 1201. kb, kilobase.

Mammalian Pro-a1(XI) Collagen
Gene(COLllAl)

17161

B

A

G G M G A C C A ~ ~ ~ T C ~ T G ~ ~ ~ C ~ W C A T C T G G G G C C A M G G T G A G A G T ~ ~ ~ T G A T1 1 1 7

34
1

3

97
3

CCA~~~TCCTCA~CC~TCGAGGCGTCCAGGGTCCCC~GTCCMCGGGGAAMCCTGGA 1 1 7 7 A G A f f i T C C T C M U ; A C C C I C A . U T C C A G ~ G G A T I C C C
3 9 3 R G P Q G P Q G P V G F P G P K G P P G
3 P G P Q G P R C V Q G P P G P T G K P G

157~~~TCGTCCAGGTGCAGAnAGGMGAGGMTGCCAGGAGMCCTGGGGCAM~217~~ G A G A T C G A C C C ~ A T G G A C I T C C G G G T C M j C C A G G T G A C A M G G T ~ G ~ T G M 1297

277~

CGAGGTCCTCMGGTCCTCCAGGTCCTCCTGGTGATGATGGMTGA~GAGMGATGGA

G

R

P

G

P

V

G

G

P

G

S

S

G

A

K

G

E

S

G

3

D

GffiAMCCTMiGCCAMGCCCACITCAGGnCGATffiCCffCCnCCCCTCCAGGTGM
7

3

G

K

P

G

P

K

G

T

S

G

G

D

G

P

P

G

P

P

G

E

(exon 9 )(exon 10)
~
~
J

K
G
R

R

G

D
G

R

R
P

P

G
Q

G

F
G

A

D
P

D

G
P

G

L
G

P
P

P

G
G
G

R
L
D

G
P
D

~
G
G

P

G

E

P

(exons l l , l 2 )
D

K

G

~

(exons 1 2 . 1 3 )
~

R

G

E

lerone 1 3 . 1 4 1

G

A

1237
413
K
4E 3

R

G

D

1351
453
G

GCCMGACCCCCCCTCCCA~AGTCG~ACCACAGGGACCMCCGGTGAG

3

G

K

T

G

P

P

G

P

G

G

V

V

G

P

Q

G

P

T

G

E

1 4 1 7 CITCCIGGTGCTGCAGGGAAMGMGGTGC~GGGTGATCCAGGTCCTCMGGTATCTCA
4 7 3 L P G A A G K E G A X G D P G P Q G I S
1477 GGGAMGATGCACCAGCAGGATIACGTGGTITCCCA~AMGAGGTCITCCTGGAGCT
4 9 3 G K D G P A G L R C F P G E R G L P G A
1537 CAGGGTGCAC~GACTGAMGGA.UGMGGTCCCCAGGGCCCACCAGGTCCAGW~C
513 Q G A P G L K G G E G P Q C P P G P V G
1 5 9 7 TCACCAGGAGMCGTGGGTCAGCAGGTACAGCn;GCCCMTTGG'TTTACGAGGGCGCCCG
533 S P G E R G S A G T A G P I G L R G R P
1 6 5 7 GGACCCICAGGGTCCTCCCCAGCTGGAGAGAMGGTGCTCCTGGAG-GGTCCC
553 G P Q G P P G P A C E K G A P G E K G

P

1717 C M G G G C C T G C A G G C A G A G A n A G T I C M G G T C ~ ~ T f f C C C A G G G C C A G C n C T
5 7 3 Q G P A G R D G V Q C P V G L P G P A G1777 CCTGCCffiCTCCCCTGGffiMGACffiAGACMGGGTCAMTIGCTGAGCCGGGAC~
5 9 3 P A G S P G E D G D X G E I G E P C Q K

697
233

1837 G G C A G C M G G G T G G C M G G G A G ~ ~ C C C T C C C G G T C C C C C A G G T ~ C M ~ A C C A
6 1 3 G S K G G K G E N G P P G P P G L Q G P

757
253

1897 G R G G T G C C C C T C G M T T G C G G A f f i T G A ~ T G M C C A G G T C f f A G A G G A C A G C A ~6 3 3 V G A P G I A G G D G E P G P R G Q Q G

817
27 3

1 9 5 7 ATGTITGGGC-GGTGATGAGGGTGCCAGAGGCITCCCTGGACCTCCTGGTCCMTA
6 5 3 N F G Q K G D E G A R G P P G P P G

877
293

P

I

2017 G G T C I T C A G f f i T C P G C C A G G C C C A C C T f f i T G - G G T G ~ ~ A T G ~ T C C A
6 1 3 G L Q G L P G P P G E K G E N G D V G P

937
313

2 0 7 7 TGCGGGCCACCCIGGTCCCICCAGCCCCCMGAGGCCCTCMGGTCCCMTGGAGCn;AT~A
6 9 3 Y G P P G P P G P R G P Q G P N G A D G

997
333

2137
713

1057
353

C
226C C C A G A T G G T G M T A T I G G A R G A T C C T M C ~ f f i ~ C A f f i A G A W C C I T C A M G T T
7 6 C P D G E Y W I D P N Q G ~ S G D S F K V

2197
733
2257
753

346C

2317
773

116C

GTMGMTITCATCATGGCCAMGGAGAMCCAGGMG~~AGTGM~MGAGG

V

R

I

S

S

W

P

K

E

K

P

G

S

W

P

S

E

F

K

R

2377
793
2437
813
2497
033

526C G C A G C C T G G T A T G A T G T C T C A T C A G G M G T I A T G A C A M G C A C I T C G ~ C ~ A T C A
176C A A Y Y D V S S G S Y D K A L R F L G S

2557
853
2617
873
2677
893
2737
913
2797
933
2857
953
2917
973
2977
993
3037
1013
46C

166C G G T A C T C A G A C C M T C C A G C C C G M ~ A M G A C ~ C M f f C A G C C A T C C T G A ~ C
5 6 C G T Q T N P A R T C X D L Q L S H P D F

FIG. 2. Nucleotide and amino acid sequences of the human COL11A1. In the first line, on the left sideof the sequence, is the numbering of the nucleotides starting from the beginning of the a-chain,while the numbersin the second line refer to the encoded amino acid residues. Note that in the C-propeptide the numbering reinitiates and itis followed by the letter C. The demarcations of the sequenced exons are indicated by the underlined
dinucleotides, and the exons are numbered 5' to 3' on the right side of the sequence. Underlined &e also theputative cross-linking sites, the C-propeptide and a-chain cystenyl residues, and the C-propeptide carbohydrate
attachment site. The arrow signifies the putative C-protease cleavage site, while the asterisk indicates the end of
the a-chain.

Mammalian Pro-al(XI) Collagen Gene (COLI l A l )

17162

A

,

0.8-

800

G
A
M CCA CCT CM GAT GOT GlT CCT CCT GAC M G CCT C M GAT GGA
Pro A l e G l y cln ASP Gly V a l ~ l cly
y &p ~ y mc l y G L U &p cly

2443
115

A
A
C
T
C
A
CAT
CCT
CCT CM CCG CCT M M CCC
CCA
M CCT GAG GCT CCC
Asp Pro Cly G l n Pro Gly ProPro
G l y Pro r.5
Gly C1u A l a Cly

a411
810

T
A
C A
G
T C
CCA CCA CCT CCT CCT =A AM CGA cm cm CCT ea m CCA CCT
Pro Pro G l y Pro Pro G l y Lym A r g Cly PI0 Pro G l y A l a AI. G l y

2398

r.5
A
G C
A
C
A
T
A
CCA CAG CCA ACA CM CCT G M AM uil Gff M G GGG G M GCA uil
Ala GluGly A r q Gln G l y Clu Ly. Gly Ala Ly. Gly G1u A l a Cly
r
.
5
ClY
T
I
G
T
G
A
T
2578
GCA G M GGT CCT
CCT
GGA A M ACC CCC CCA CK CCT CCT
CAG
Gw
860 AI.
c1u cly pro pro c l y L ~ Smr c l y pro v.1 G I pro~
GI" c l y
T C

2533
845

T T
2623
875

M GCA CCA M G CCT CCT CCA G M CCT Cl7 CGG GGC A K CCT CGT
Pro A h Gly Lys Pro Cly Pro Clu cly U u Arq Cly 11. Pro G l y

1661
890

M C l G CCA G M CM CCT CK C f f GGA GCT CU CCC CM GAT GGA
Pro V a l Gly C l u G l n G l y U u Pro G l y A l a A l a Gly Cln Asp Cly

2113
905

CCA M CCT CCT ATG CCA CCT M GGC T T A M CCT CK AM CCT
Pro Pro Cly Pro Pl.t Cly Pro Pro cly U u Pro G l y UYLy. CLy

a758
920

GAC ccr ccc KC MG

c

C

1

r

8 0.6"
0.5".

A

n

0

1

T

B

C

nn

z 0.4"a
E 0.3-.

p0.2".

a 0.1-.
0
0

A

r.5

0.7-.

1
10

1
1
I
20
30
40
FRACTION NUMBER

I
50

'

FIG.4. CM-cellulose chromatography of Type XI heat-denatured chains. Brackets indicate the fractions pooled for further

analysis (A, B, and C).
start
The
the arrow.

of the NaCl gradient is signified by

U Y

T

2803
935
2141

950
2893
965
a931
980

B

A.p

T

Pro Cly r.5

A
CCT GM MC CCA CAT cm CCT TTA ATT ccc
Ly. G l y G I " Lys Cly H i s Pro G l Y U u 110 G l y

C

G

A
T
CTG A T T CCT CCT
CCA
GGA G M C M GGG G M AM CCT CAC CW, GGG
U u 11. cly Pro Pro G l y Clu G l n cly C I Y Lys Cly Asp A r q G l y

2
"
h
.
L
-

1
G
C
T
C
T
C
CK M GGA ACT CM GGA M CU GGA GCA A M GGG CAT WC CCA
U u Pro Gly Thr G l n Cly 1.5 Pro Gly AI. Ly. G l Y ASP G l Y C l Y
C
A
A
T
A
ATI' M CCT CCT CCT CCT CCC TTA CCT CCA
CCT
uil M CCA CCC
11. Pro Cly Pro A l a Cly Pro U Y Cly Pro Pro C l Y Pro Pro G l Y
11.
A
T
GCT
T
G
TTA CCA CCT CCT CM ccc CCA MG CCT MC AM GGC M ACT CCA
U u Pro G l y Pro Cln Cly Pro Ly. Cly An.
Ly. Cly I.
5
Thr Gly
Ala
5.1

3021
1010

TA
G
A G
A
CCCGCl't3CCCAGAMCCTGACACTCCTCPTCUGGGMMGC-S
R O A l a G l y G l n Lye Cly Asp r.5
G l y U u Pro G l y Pro Pro Cly
mr
.M t
T
A
*
A
A
T
T
M CU CCT CCA M CCT G M Gtc A ' I T CAG
CCT
T T A CCAATCTIC
Pro Pro ClY Pro PI0c1y GI" V a l 11. Cl" Pr'o U" Pro 11. Lo"

22C
IC

TCC K C AM AM ACG AGA AGA CAT A f f G M CCC A% C M CCA GAT
5.1' r.S
Ly.
Ly.
T
h
l A r q A r q HI. Thr CLU C l y Mat Cln A l a A8P

67C
23C

CU GAT GAT M T A T T CPT CAT T I C lXX GAT GGA A l G G M G MATA
A l a A'p Anp A,"
11. U Y A.p Tyr r.S
Amp Cly Mat Glu Clu 11.

112C
38C

TI7 GGT TCC CK M T K C CTG AM C M GAC A X GAG CAT A l G AM
Cly r.5
U u A m 5.1 U u Ly. Cln Asp 11. Clu His Mst Lym

2981
995

S B C A S

A C G

C.

A

A

C

PC0
C
ClY

Ph.

C

A

C'C

11.

r.5
C

C

C

T

G

I

C

G

FIG.5. 6% SDS-PAGE of the Type XI chains. The purified
Type XI collagen is in the lunes labeled S whereas the separated
chains, pooled after CM-cellulose chromatography, are labeled as inFig. 4 as A, B, and C.

aoac
68C
247c
13c
292c
98C
337c
ll3C

mac
128C
4a7c
143C

C

517c
113c
562C
188C
607C
ao3c

652C
218C
697C
133.2
742C

mc

AI.

717C
263.2

CY.

T
L C
C I T U G M T C A G M G " C 0 0 1 ~ C M G U ~ M ' 7 l T W T l T~ m pGI" ~ m GI"n
LY. m. GIY ~u G ~ Uv.1 GLY RO v.1
R.

CR Gu: T U
U U Gly X

FIG.3. Comparison between the human and rat sequences.
Numbering of the human sequence is as in Fig. 2. Only the nucleotide(above) and amino acid (below) differences with the rat sequence areindicated. The asterisk signifies the end of the cr-chain, while thearrow indicates the putative C-protease cleavage site.

Biochemical Analyses of the Human al(XI) Collagen
Chin-Identification of the collagen precipitating at 1.2 M
NaCl as Type XI collagen was accomplished by gel electrophoresis, in which the al(XI), a2(XI), and a3(XI) chainsappeared as bands of approximately equal intensities (Fig. 5 ) ,and by the chromatographic elution of the chains on a CMcellulose column (Fig. 4). The separated chains were further
characterized by amino acid analyses (data not shown), andelectrophoresis of their CNBr derived peptides (Fig. 6). By allthese criteria, our preparation was identical to the one originally described by Burgeson and Hollister (1979). The CNBrderived fragments of the al(X1) chain were then separatedby HPLC (Fig. 7). All peptide-containing fractions were analyzed bygel electrophoresis and amino acid analysis. Two
fractions containing short fragments undetectable in 15%
acrylamide gels ( A and B in Fig. 7), and one fraction containing a 35-kDa peptide (C in Fig. 7) were purified by a second
HPLC run. The fractions indicated by arrows in Fig. 8 werethen selected for sequencing. The amino acid sequences of
peptides A, B, and C are shown in Fig. 9 together with thetranslated amino acid sequence of the cDNA clones. This
comparison conclusively proved that the clones code for thep r o d chain of Type XI collagen. Incidentally, similar analyses using a l ( V ) CNBr peptides and tryptic peptides encompassing a total of 136 amino acid residues revealed a 86%
sequence similarity with the d ( X 1 ) chain (data not shown).
Genomic Cloning-One of the most distinctive features ofMammalian Pro-al(XI) Collagen Gene (COLllAl)

17163

B C A I I

I

Q

0

10

20

30

40

50

60

70

60

70

60

70

TIME (Minutes)

0

10

20

30

40

50

TIME (Minutes)
FIG. 6. 15%SDS-PAGE of the CNBr derived peptides ofa2(I);
the purified Type XI chains. Lane I,Type I collagen (al(I)Jnlune B, al(X1); lane C, a2(XI); lune A, a3(XI); lune II, al(I1).
1.0

C

3 0.8
C

*Z; 0.6
Y

W

0.4

0

2

20

30

40

50

TIME (Minutes)

8 0.2

FIG. 8. Second reversed-phase HPLC of the CNBr-derived
peptides from the al(X1) chains. The fractions labeled A, B, andC in Fig. 7 were eluted with the same gradient as in Fig. 7, exceptthat 9 mM trifluoroacetic acid was used. The arrows indicate thefractions that eventually were sequenced.

v)

$

10

0

0

10

20

30

40

50

60

70

TIME (Minutes)

P E P T I D E A: RGEDGEIGPRGLPGEAGPRGLLGPRGXPGAPGQ
cDNA: RGEDGEIGPRGLPGEAGPRGLLGPRGTPGAPGQ
108
140

FIG. 7. Reversed-phase HPLC of the CNBr-derived peptides from the al(X1) chain. Peptides derived from 1 mg of al(X1)chain were eluted with an aqueous gradient of acetonitrile (12.444.4%) over 60 min, in the presence of 10 mM heptafluorobutyricacid. Brackets indicate the fractions that were used for further analysis (A, B, and C).

P E P T I D E B: GPQGEPGPPGQ&NPGP&LPGP&PIGPPGEXGP&KPG
cONA: GPQGEPGPPGQQGNPGP@iLPGPQGPIGPPGEKGP@KPG
157
196

the collagen genes is the pattern of distribution of the exons
coding for the triplehelical domains (for review
asee Ramirez
et al., 1985). This feature can in fact be used to divide therelatively large group of collagen genes into distinct subfamilies with identical exon/intron arrangements. Accordingly,
characterization of the Type I, 11,111, and V collagen genes
has provided a basis for categorizing these collagens in a
distinct subfamily, the fibrillar collagens (Ramirez et aL,
1985). To examine whether also COLllAl belongs to thisgroup, we isolated several genomic clones from a humanlibrary and analyzed the structure of one of them, Pen 3.
Because of the size and complexity of COL11A1, rather thandetailing the organization of the entire clone, we decided tocharacterize a selected portion by shot-gun subcloning andsequencing. To this end, the EcoRI-digested DNA of Pen 3

FIG. 9. Sequence comparison between three al(X1) peptides
and the translation of the human cDNAs. Numbering of theamino acid residues is asin Figs. 2 and 3. Undetermined amino acids
are labeled X.Note that prolines in the third, Y , position are hydroxylated.

P E P T I D E C: GLXGDRGEVGQIGPRG
cDNA: GLKGDRGEVGQIGPRG

28

268

3

was hybridized to theB’-most XhoI subfragment of OK4 (Fig.
1). The positive 5.5-kilobase EcoRI fragment of Pen 3 wasinserted into pUC18, and in turn used to generate a collectionof Sau3A subclones which were then screened with the original XhoI probe of OK4. Twenty-four of the positively hybridizing Sau3A subclones were randomly chosen and sequenced.
This led to the identification of seven triple-helical domaincoding exons, whose relative location with respect to thecDNA sequence is shown in Fig. 2. Several points should be
noted. Firstly, the exons cover a continuous stretch of 360

Mammalian Pro-a1(XI) Collagen Gene(COL11Al)

17164

TABLEI
Sequence divergence between fibrillarcollagen genes and COLlAl
Amino acids

Nucleotides%

40

COL2A1COLlA2
COL3AlCOL5A2COLllAl

33
39
40
48
62

28
35
42
52

TABLEI1
Codon usage in the triple helical domains of the fibrillar collagen
genes
Computations of the data were based on theentire sequence of thetriple-helical domains of COLlAl (Bernard et al., 1983a; D’Alessio etal., 1988), COLlA2 (Bernard et al., 1983b;de Wet et al., 1987),
COL5A2 (Weil et al., 1987; D. Woodbury, V. Benson-Chanda, and F.
Ramirez, manuscript in preparation), COL3A1 (Chu et al., 1985; R.
Janeczko and F. Ramirez, manuscript in preparation), and COL2Al(Sangiorgi et al., 1985; M. W. Su, V. Benson-Chanda, B. Lee, H.
Vissing, and F. Ramirez, manuscript in preparation).
Amino Third COL- COL- COL- COL- COL- COL-

lishing the patterns of codon usage, an indicator of kinshipamong related genes.
Although pairwise comparison of the C-propeptide sequences showed minimal degree of homology, with the exception of few nucleotides around the conserved cysteinyl residues (for a review, see Dion and Myers, 1987), it enabled us
to position COLlAll closer to COL5A2 and at the highest
point of divergence from COLlAl (Table I).
The analysis of the wobble base conservation in the triplehelical domain codons established a kinship among COL3A1,
COL5A2, and COL11A1. This conclusion rests on the findingthat in COLlAl, COLlA2, and COL2Al there is a strong
preference in the wobble position first for T and secondly forC, while in COL3A1, COL5A2, and COLllAl the first preference is for T, and secondly for A (Table 11).
DISCUSSION

Identification of the Pro-al(XI) CollagencDNAClonesThe isolation of several cDNA clones from two mammalian
species provides the first detailed study of the primary structure of a Type XI collagen chain. The overall length of the
acid
base
1Al
2A1
1A2
3A1
5A2
llAl
human cDNAs is nearly 5 kilobases, and, in addition to the%
3’-untranslated region, it comprises the entireC-terminal
Glycine
T
52
43
54
4445
37
propeptide and, in our estimate, 99% of the triple-helical
C
27
28
12
20
16
13
domain. The identity of the recombinant molecules was esA
18
24
22
37
34
28
tablished by direct comparison with the amino acid sequence
G
3
5
4
7
13
14
of three al(X1)CNBr-derived peptides determined by Edman
T
59
61
63
55
51
51
Prolinedegradation (Fig. 9). Surprisingly, our experiments indicate
38
28
21
13
9
7
C
that COLllAl expression, albeit at extremely low levels, is
A
3
11
15
31
39
33
not confined to cartilagenous tissues. It remains to be demG
O
0
1
1
7
3
onstrated whether such a finding is indicative of post-tranAlanine T
75
59
77
61
50
44
scriptional controls that restrict Type XI
biosynthesis in some
11
29C
20
15
14
6
A
5
12
8
23
33
45
non-cartilagenous tissue, such as placenta, or whether, given
G
O
0
0
2
11
0
the low representation of the mRNA, the protein is undetectable by conventional methods. Notwithstanding this problem,thesestudieshave generated a number of important pieces ofnucleotides with a base pair size pattern, 54-54-54-54-45-5445, clearly indicative of a fibrillar collagen motif. Furthermore, information related to the structure and evolution of two of
this particular pattern is found only in one specific region of the major domains of the procollagen molecule, the C-propepthe Type I genes, namely between exons 9 and 15 (ascounted tide and the triple helix.
C-propeptide-It is now widely accepted that intracellularfrom the 5’ to 3’ end) (de Wet et al., 1987; D’Alessio et al.,foldingof the procollagen chains into the triple helix begins
1988). These exons code for amino acid residues 37-156.
Within this segment of the triple helical domain lies a cross- at the C-propeptides with the formation of inter- and intralinking site, whose sequence, Gly-X-Hyl-Gly-His-Arg, and chain disulfide bonds (Uitto and Prockop, 1974; Rosenbloom
relative location, between residues 85 and 90, is highly con- et al., 1976).Less understood is how this process is selectively
served among the fibrillar collagen molecules. Moreover, this guided in those instances where either several different colcanonical element is the last hexapeptide encoded by exon 11, lagen types are co-expressed in the same tissue, or the same
which is 54 bp in size (de Wet et al., 1987; D’Alessio et al., type displays a molecular heterogeneity in different tissues
1988). Inspection of the translation product of the al(X1) and/or at different times of development (Miller and Gay,sequence identified the hexapeptide Gly-Asp-Lys-Gly-His- 1987). With the increasing acquisition of structural informaArg within the last 18 bp of a 54-bp exon (Fig. 2). Hence, by tion, several investigators have begun to analyze the data inanalogy to other fibrillar collagen genes, this exon was num- search of evolutionary conserved features that may shed newbered as 11 (Fig. 2). This conclusion enabled us to extrapolate light on this problem (Showalter et al.,1980; Fuller andthat our sequence begins at the 13th amino acid residue of Boedtker 1981; Bernard et al., 1983a, 1983b; Yamada et al.,the triple helical domain and that the al(X1) chain is 1017 1983; Ninomiya et al., 1984; Sandell et al., 1984; Sangiorgi etal., 1985;Myers et al., 1985;Chu et al., 1985).One such feature
residues long (Fig. 2).
Sequence Comparison between the Pro-al(XI) Chain and is the number and location of the cysteinyl residues in the Cthe Other Fibrillar Collagens-Having categorized the pro- propeptides of the fibrillar collagens (Dion and Myers, 1987).
al(X1) chain as a member of the fibrillar-forming collagens, Two distinct patterns emerge. In polypeptides that form howe then attempted to more firmly place this gene within the motrimers (al(II), al(II1))or both homo- and heterotrimersgroup. To thisend, we analyzed the human C-propeptide and (al(I)), 8 cysteinyl residues are present, whereas in procollatriple-helical domain sequences. Of these two segments, the gen chains that only form part of heterotrimers (a2(I) andformer, which lacks the repetitive Gly-X-Y triplet structure, a2(V)), 7 of these residues are found. Based on studies of thehas been used for ascertaining the level of sequence similarity avian al(1) and al(I1) collagens, cysteines 5-8 (numbering
between each of the collagen genesand COLlAl.On the other from 5’ to 3’) are involved in intrachain linkages and are
hand, the abundance of glycine, proline, and alanine residues invariable in number (Olsen, 1982). In point of fact, in a
in the triple-helical domain has served as a basis for estab- naturally occurring variant a mutation affecting the last ofMammalian Pro-al(XI)Gene
Collagen(COLllAl)

17165

the a2(I) cysteinyl residues prevents heterotrimer assembly could reflect the existence of different collagenase specifici(Pihlajaniemi et al., 1984). On the other hand, the absence of ties. Such a notion is supported not only by the isolation of aeither cysteine number 2 (cuP(1)) or number 3 (a2(V))may be Type V-specific collagenase (Mainardi et al., 1980), but alsoa consequence of chain-specific requirements for those chains by the work of Fields et al. (1987) which showedother possiblethat associate exclusively in heterotrimeric forms. This idea cleavage sequences for the enzyme, and emphasized the imseems to be further supported by the finding that in both the portance of secondary and tertiary structural considerations
human and rat pro-al(X1)cysteine number 2 is missing, and in addition to sequence specificity.
A final feature, which seems unique to the al(X1)chain, isthat, like in a2(I) and a2(V), thisis due to a single nucleotidechange from a cysteine to a serine codon (Fig. 2). It would be the presence of an unusual cysteinyl residue located approxiof interest to determine whether the absence of a potential mately in themiddle of the triple-helical domain. This residue
interchain disulfide linkage is indeed a feature of all hetero- is not utilized in the formation of an interchain disulfide
trimeric collagen chains (&(XI) and a3(V)), and conversely bond, because reduction of the pepsin-treated molecule does
whether the presence of all 4 cysteines (1 to 4) is always not affect the migration of Type XI chains on SDS-PAGE
related to homotrimer-forming chains (al(V)). The idea is (Burgeson and Hollister, 1979; Kielty et ul., 1984). Therefore,al(I1) we speculate that this residue may be involved in bonding toalso consistent with the hypothesis that the a3(XI) andchains are productsof the same gene, since al(II), like al(I), other components in the extracellular matrix.
Type XI, a Fibrillar Collagen-Our structural findings
has 8 cysteinyl residues and is capable of both homo- andclearly define Type XI asa fibrillar collagen. This conclusion
heterotrimeric assembly.
A second feature that places the pro-al(X1) chain in the confirms the previous classification of the molecule on thebasis of protein data (Miller and Gay, 1987).Although limitedgroup of fibrillar collagens is the presence of a tripeptide(Asn-Phe-Thr), which is a potential acceptor for N-aspara- to a very small portion of the gene, our characterization ofginyl-linked carbohydrateattachment in the C-propeptide COLllAl strongly suggests that its organization is identicalto thatof other fibrillar collagen genes.
(Struck and Lennarz, 1980) (Fig. 2).
Analyses of the C-propeptide and helical domain sequences
Finally, in the human sequence two possible C-proteinasecleavage sites were identified at C21-C22 and C23-C24 (Dion showed that Type XI exhibits a close kinship with Types I11
and Myers, 1987). Because the rat chain lacks the latter, we and V. The kinship with Type V was not surprising because
have assigned the cleavage site to the Ala-Asp sequence of of the already suggested structural and, possibly, functional
the former (Figs. 2 and 3). Assuming that our premise is similarities between the two minor collagens. In addition, andin line with what has been seen for most of the fibrillar
correct, the C-terminal telopeptide is then21 residues long.
Helical Domuin-The fibrillar nature of the pro-al(X1) collagen genes, the COL11Al offers another example of chrochain has been confirmed by the inspection of the triple- mosomal dispersion, for it resides on the short arm of chrohelical domain sequence, whose long uninterrupted motif of mosome 1 (Henry et al., 1988).
Although the data presented here quite convincingly place
Gly-X-Y repeats is the most obvious evidence. In addition,
the presence of the cross-linking sites further strengthens thisType XI among the fibrillar collagens, it will be important toargument. In the fibrillar collagens the lysines involved in further detail theorganization of COLllAl in order to identhese intermolecular cross-links are located at positions 87 tify possible deviations from the prototypical structure of aand 930 of the triple helix and in thetwo telopeptide domains fibrillar collagen gene. Fusion of two triple-helical domain(Eyre etal., 1984). In the al(X1) sequence, three of the coding exons in COLlAl andvariations in the number of Nconserved lysines were identified. Two of them lie within the propeptide encoding exons in COL2A1 and COL3Al have infact been observed (Chu et al., 1984, 1985).3. These findings
helix, one at position 87 and the other at 927. The latter
havebeen helpful in tracing the evolutionary history of therepresents a positional variation as compared to themajority
genes,
as well as delineating some of the selective pressuresof the other fibrillar chains. Suchan exception is notthe firstone, as this cross-linking site is located at position 933 in the exerted upon their products. Finally, and relevant to thea 2 chain of Type I collagen (Eyre et al., 1984; de Wet et al., surprising finding of pro-al(X1) mRNA in cells of non-carti1987). The C-terminal telopeptide in all but two of the colla- lagenous origin, the analysis of potential regulatory elements
gens (a2(I) anda2(V)) contains the forth cross-linking lysine, should enhance our understanding of Type XI function in
9 residues upstream from the C-protease cleavage site (Eyre physiological and pathological conditions.
et al., 1984;Weil et al., 1987;de Wet et al., 1987). In the
Acknowledgments-We wish to thank Drs. Berg and Okayama foral(X1) two consecutive lysines are seen in both mammalian providing
us with the cDNA library, Dr. B. Brodsky for many helpful
species, 11 and 12 amino acid residues upstream from the C- discussions and suggestions during the courseof this work, E. determinalend of the telopeptide. Whetherthese twolysyl
Miguel and E. Wan for their skillful technical assistance, and Roresidues do indeed participate in the cross-linking processes seann Lingeza for preparing and typing the manuscript.
must await experimental confirmation.
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17166

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