THE JOURNAL OP BIOLOGICAL CHEMISTRY
0 1990 by The American Society for BiochemistryVol. 265, No. 11, Issue of April 15, pp. 6423-6426,199O

Printed in U.S.A.

and Molecular Biology, Inc

Pro-a 1 (XI) Collagen
STRUCTURE OF THE AMINO-TERMINALCELL LINES*

PROPEPTIDEAND EXPRESSION

OF THE GENE IN TUMOR(Received

Hidekatsu

YoshiokaS

From the Department
Health Science Center,and Francescoof Microbiology
Brooklyn,for publication,

November21, 1989)

Ramirezg

and Immunology,
New York 11203

Morse

We have determined
the nucleotide
sequenceof several overlapping
cDNA
clones encoding
the aminoterminal
portionof human(Al
procollagen.
These
experiments
have revealed
that this domain of the proa(X1) chain
displays
structuralfeaturescommontoother fibrillar
procollagenmolecules,
such as a putative amino-terminal
proteinasecleavagesite and aninterrupted
collagenous
segment.
In the latter,
structuralsimilarities
were noted when al(X1)
was comparedwithal(I1)
and a2(V)procollagens.
Overall,
however,
the amino-terminal
region of pro-crl(X1)differs greatly
in compositionand size from that of otherfibrillar
chains. Nearly
three-fourthsof this domain
isin fact composedof a 383-aminoacid globular
regionin which a 3-cysteine
clustersignals the transitiontoa long and highly
acidic carboxyl-terminal
segment.
Finally,
the unrestricted
expressionof this cartilagespecificcollagengene has been confirmedby the finding of high levels of pro-crl(X1)mRNA
in two humanrhabdomyosarcoma
cell lines.

Type XI collagen is a minor extracellular component ofhyaline cartilage (1). This macromolecule is composed of three
distinct subunits, termed oll(XI), a2(XI), and (~3(X1) collagens (2). The genes that code for the precursor procollagenform of (Aland cuB(X1)chains (COLllAl and COLllA2)’
have been recently cloned and localized on human chromosomes 1 and 6, respectively (3-5). In contrast, the (~3(X1)chain is believed to be a post-translational variation of the
major cartilage collagen, (Al(6). Aside from inferring someof the primary structure of pro-cyl(X1) and pro-cyB(XI) chains,the cloning experiments have also indicated that type XI
collagen belongs to the fibril-forming group of molecules
which, in addition, includes types I, II, III, and V (3, 5).
* This work was supported
from Grants
AR-38648
and HD-22657
from the National
Institutesof Health.
The costs of publicationofthis article
were defrayedin part by the paymentof page charges.
This
articlemust therefore
be hereby
marked“advertisement”
inaccordancewith 18 U.S.C. Section
1734 solely to indicate
this fact.
The nucleotide
sequence(s)reportedin this paper ha.s been submitted
to the GenBankTM/EMBL
Data
Bank
with
accession
number(s)505407.
$ On leave of absence from Kumamoto
MedicalSchool, Kumamoto(Japan).
§ To whom
correspondence
and reprint
requestsshouldbe addressed.
Present
address:
Brookdale
Centerfor Molecular
Biology,
Mount
Sinai School of Medicine,
1 Gustave
Levy Place, New York,NY 10029.
1 The abbreviations
used are: COLllAl,
the gene encoding
proal(XI)
collagen;
COLllAQ,
the gene encoding
pro-aL(X1)
collagen;N-propeptide,
the amino-terminal
propeptideof the procollagenmolecule; N-proteinase,the enzymethat cleaves the N-propeptide.

Instituteof Molecular

Genetics,

State

Universityof NewYork,

Furthermore, these studies have revealed that, unlike
COLllA2, COLllAl is also expressed in noncartilagenous
tissues, for pro-cul(X1) clones were isolated from cDNA libraries prepared from placental tissue and a SV40-transformed fibroblast cell line (3, 5).
Emerging biochemical evidence suggests that collagen XI
may play an important role in fibrillogenesis by controlling
lateral growth of collagen II fibrils (2, 7). In non-cartilage
tissue, a comparable function, with respect to collagen I fibrils,
has been proposed for the minor collagen V (8). This, andadditional structural and metabolic similarities between collagens V and XI, have lent support to the notion that therelationship between collagens II and XI in cartilage is analogous to that of collagens I and V in other tissues (9).
Moreover, the recent finding in adult bovine bone of crl(XI),
al(V), and (Ye chains in the ratio of 1:l:l has suggested
the possible formation of cross-type heterotrimers between
some of the subunits of collagens V and XI (10).
To gather more information about the structure and expression of this minor cartilage collagen type, we have here
extended our cloning experiments on pro-oll(X1). This study
has inferred the organization of the N-propeptide and thus
completed the determination of the amino acid sequence ofthe human chain. Moreover, analysis of a large panel of celllines has confirmed the unrestricted expression of the geneby documenting high levels of pro-al(X1) mRNA in two
human tumor cells of noncartilagenous origin.
MATERIALSAND

METHODS

cDNA
Cloningand DNA
Sequencing-Primer
extensioncDNA
cloningusing the hgtl0 phage vector
and screeningof the recombinants, as well as isolationand purificationof the resulting
positiveclones, were essentiallyas previously
described(11). Sequencing
wascarriedout according
to the dideoxy
chain termination
procedureondouble-stranded
DNA (12). Multiple
sequencing
of both strands
wasperformed
for the sequencesof this report.
Computeranalysiswasdone using the sequence
analysisprogramdevelopedby Mount
andConrad(13).
RNA Isolation
and Northern
Blot Hybridization-For
Northernblots, poly(A)+RNA was purified
from cultured
cells using the isothiocyanate
methodfollowedby elution
throughan oligo(dT)-cellulose (Collaborative
Research)column(14). Approximately
1 pg ofpoly(A)+RNA was electrophoresed
in 0.8% gel in the presenceofformamide/formaldehyde,transferred
onto nitrocellulose
filters (Millipore),and hybridized
to nick-translated
DNA as described(15).
The 14 cell lines used for the Northernblot experiments
are listed
below along with the source from which they were obtained.
AmericanType Culture
Collection:
HT-1080(CCL 121), humanfibrosarcoma;
RD (CCL 136), humanembryonal
rhabdomyosarcoma;
A-673 (CRL
1598) and A-204 (HTB82), humanrhabdomyosarcomas;
Hs 578T(HTB126), humanbreastductalcarcinoma;
KATO
III (HT 103),
human
gastric carcinoma;
A-549 (CCL 185), humanlung carcinoma;
Hs 696 (HTB
151), primaryhuman
adenocarcinoma
metastatictobone sacrum;
CRL
1183, humanskin fibroblasts;
AV, (CCL
21),

6423

6424

Structureand Expression of Type XI Collagenhuman amnion cells. National Institute of General Medical Sciences:
GM00637D, SV40transformed
human adult fibroblasts. Dr. M.
Young (National Institute of Dental Research, Bone Research
Branch): 563 and 564, bovine fetal long bone; 590, human adult bone.
RESULTSANDDISCUSSION

Oligomer-specific cDNA Cloning-We have previously isolated and sequenced two overlapping
human cDNA clones,
OK4 and Hpl 1, coding a 264-amino acid C-propeptide and1005 amino acid residues of the triple helical domain of humanpro-a(X1) collagen (3). To complete the determination of the
primary structure of pro-al(XI), we have now extended oursequence analysis by oligomer-specific cDNA cloning.
To this end, a synthetic oligomer, termed lA, complementary to a sequence located near the 5’ end of clone Hpl 1, wasused to prime reverse transcription of pro-al(X1) mRNA
(Figs. 1 and 2). To ensure specificity, the resulting clones
were screened with an oligomer probe, termed lB, positioned
7 nucleotides 5’ of the sequence of oligomer 1A (Figs. 1 and
2). As a result, four positive recombinants were identified andcharacterized. Upon sequence inspection, the largest of them,
HY3, was found to contain an open reading frame whose
conceptual translation overlaps with the sequence of Hpl 1and extends into a noncollagenous domain (Fig. 1). Hence, asecond cloning experiment was performed using oligomer 2Afor RNA priming and oligomer 2B for screening of cDNAs(Figs. 1 and 2). This led to the isolation of two short clones
the longest of which, HY93, extends nearly 150 base pairs 5’of HY3 (Fig. 1). Moreover, parallel probing of the oligomer
la-primed cDNA library with oligomer 2B identified a 1.3kilobase pair cDNA, HY477, which overlaps with Hpl 1 andextends an additional 280 base pairs 5’ of HY93 (Fig. 1).
Since HY477 also bears a continuous open reading frame,oligomers 3A and 3B were synthesized and used for primer
extension cDNA cloning and for screening of the recombinants, respectively (Figs. 1 and 2). Sequencing of two of theresulting positive clones, HY81 and HY83, established theiridentity and, more importantly, revealed the untranslated
nature of the first 161 base pairs of the longest clone, HY83

HY3
HY93
HY477

H
HY80

I

I

l-1

I

I

HYSO

H
1 kb
I

-1

An
overall restriction map of the cDNAs generated in this study is shownin relationship to the relative position of the signal peptide (whitebon), N-propeptide (hat&dbox), and helical domain (black box) of
the protein. The dotted line signifies noncoding sequences, the lettersthe following enzymes: ApaI (A), BaZI (B), EcoRI (E), NcoI (N), and
PstI (P). The arrows indicate the positions of the oligomers used for
RNA priming and library screening, and the asterisk the 5’ foremost
position of the previously described clone Hpl l(3). Below the map
are the relative positions of the cDNAs generated in the primer
extension cloning experiments.
FIG.

1. Characterizationofpro-al(X1)cDNA

clones.

(Figs. 1 and 2). Confirmatory of these findings was the independent isolation of two very short cDNAs, HY50 and HY80,
from the oligomer lA-primed library probed with the 5’ EcoRI
fragment of clone HY83 (Fig. 1).
Aside from using distinct oligomers for priming and screening, other potential cloning artifacts were excluded by comparing the Northern blot hybridization pattern of the original
pro-cYl(X1) cDNA, OK4, with those of two primer-extended
recombinants, namely HY93 and the 5’ EcoRI subclone of
HY83 (Fig. 3).
Sequence Analysis-A composite of the nucleotide sequenceof the cDNA clones that cover the 5’ portion of the proal(X1) collagen mRNA is shown in Fig. 2 along with its aminoacid translation.
Following the ATG codon is a stretch of 36 amino acids
that displays some of the features of a signal peptide (16).
Specifically, a positively charged residue, Arg, precedes ahydrophobic core that is in turn followed by a large polaramino acid, Gln, positioned 8 residues to the left of a putative
cleavage site, Ala-Ala (Fig. 2).
Four-hundred ninety-two amino acids separate the putative
signal peptidase cleavage site from the beginning of the triple
helical domain of the al(X1) chain. This represents the longest N-propeptide hitherto described in a fibrillar procollagen.
Moreover, the structure of the al(X1) N-propeptide is highly
divergent from those of other fibrillar molecules. In broad
terms, a fibrillar procollagen N-propeptide can be generalized
as displaying either a two-domain or a three-domain configuration (17). The latter consists of a globular region with 10
characteristically spaced cysteines that are believed to be
involved in intrachain bonding, a continuous or interrupted
collagenous stretch that participates in triple helical formation, and a short nonhelical segment that connects the amino
peptide to the central triple helical domain. All but the globular region are present in the alternative two-domain structure (17).
Upon coarse examination, the pro-cYl(X1) N-propeptide
appears to be divided into three consecutive elements: a longglobular domain, an interrupted collagenous region, and ashort nonhelical segment. The globular region is extremely
long, for it encompasses 383 amino acids or nearly threefourths of the N-propeptide, and displays a unique arrangement of sequences. To be precise, 3 cysteines positioned
almost in the middle of the globular region demarcate a sharptransition from a slightly basic into a highly acidic region
with theoretical p1 values of 8.98 and 3.61, respectively (Fig.
2). Although the significance of such an unusual architecture
cannot be inferred from the sequencing data, one may speculate that it might be involved in the slow, and probably
partial, proteolysis of type XI procollagen to collagen and/or
in interactions with other matrix components (2). Antibodies
produced on the basis of the data gathered from this study
are currently being utilized to elucidate this and other important questions that pertain to collagen XI metabolism andfunction.
Twenty-four Gly-X-Y repeated triplets constitute the collagenous segment of the pro-al(X1) N-propeptide. Two noncollagenous interruptions divide the repeated motif into threeclusters of 4, 17, and 3 Gly-X-Y triplets (Fig. 2). We argue
that, because of its length and the low imino acid content, it
is most unlikely that the last set of repeats can participate intriple helical assembly. With the exception of the length of
the noncollagenous interruptions, 10 versus 4 residues, theorganization of the first two collagenous clusters of proal(X1) resembles somewhat those of the pro-cYl(I1) and procyB(V)N-propeptides (18, 19). More interestingly, the threeStructureand Expression of Type XI Collagen

6425

FIG. 2. Nucleotide
and deduced
aminoacid sequencesof the 5’ portionof pro-cr(X1)collagen.
Capitalletters indicate
the coding sequences,whereaslower case letters signify
the 5’ noncoding
sequences.
Amino
acid
residues
are numbered
above the sequences.
Numberingof the N-propeptide
sequence
begins immediately
after
the putative
signal peptide
cleavagesite and ends at the beginningof the triple helical domain(double verticalbar). The triple helical domain
sequences
are numberedfrom the first Gly and extend
to the sequenceof Hpl 1(asterisk)
used for the generationof the first sets of synthetic
oligomers.
Dotted lines above the nucleotide
sequence
indicate
the positionsof the synthetic
oligomers,
which are from 3’ to 5’: lA, lB, 2A, 2B, 3A, and 3B. Cysteinesof the globular
region are boxed. Bordersof the collagenous
segment
are signified
by the symbol(A), while its
interruptions
are underlined.
Arrowsindicate
the putative
signal peptidase
and N-proteinasecleavagesites.
Lv
al(=)

1:
I

*6.3*
2

**

*

*

(8)

3

al(n)
a2 w
al(n)
,-(I)
al(Irr,@l(I)4blot hybridizationsof pro-crl(X1)
cDNAs.
The probes used are OK4 (lanes 1 and 3), HY93 (lane 2), and the 5’
EcoRI
fragmentof HY83(lane 4). Approximately
1 pg of A-204
poly(A)+RNA was used in each of the two Northernblots. Sizes ofthe pro-cYl(X1)mRNA
species are indicatedin kilobases.

Gly-X-Y repeats to the left of the first interruption score anearly perfect match when the three human chains are aligned
together (Fig. 4A). We believe that these structural similarities are consonant with biochemical evidence strongly suggesting the formation of cross-type heterotrimers between thesubunits of types V and XI, as well as with the notion of acommon subunit between collagens II and XI (6, 7).
Two important structures are usually found in the shortnonhelical segment which connects the collagenous region of
the N-propeptide to the a-chain: the N-proteinase cleavage
site and the cross-linking lysine residue (17, 20). While thelatter is absent in pro-cul(XI), a potential N-proteinase cleavage site, Ile-Ser-AlaJGln, is recognized on the basis of its
similarity to the moderately conserved sequence of fibrillar
procollagen chains (Fig. 4B) (19). Assuming that our premise
is correct, the (Alchain then contains a 17-amino acidAla n-c ay

l

al(n)

@

FIG. 3. Northernay,x2(“)I

Gl" Lys cay an
Al?.&

I

-

*

11eserAla
me*AlameAla-

1 can
11 1 -

2z?.Ya~:I_

Lys my all
_

_

-

PIU
-

l
GlyLe”“alplv
ay
Asp

*

l
ne

Ala

“al

“al

Lb-5

ASD

ne

Gin
-

mr
“al

FIG. 4. Sequencecomparisonof selected
portionsof humanfibrillar
collagenN-propeptides.
Punel A, collagenous
segments
with the adjacentcollagenousinterruptions
are underlined.
Panel B,sequencearound the N-proteinasecleavagesite (arrow).
Dashes show
no change from the pro-cul(X1)sequence,
whereasasterisk
indicate
gaps inserted
to give best alignment.

amino-terminal telopeptide which, unique among the fibrillar
collagens, lacks the cross-linking site.
COLlAll
Expressionin Noncolhgenous
Tissues-The isolation of human pro-cYl(X1) clones from placental tissue andSV40-transformed fibroblast cDNA libraries has previously
inferred an unrestricted expression of COLllAl (3). In contrast, Northern blot analysis has more recently shown that
COLllA2 is not expressed in human adult liver, skin, andtendon (5).
To further explore this phenomenon, we analyzed the collagen content in the RNAs from a large panel of normal andtumor cell lines with probes coding for pro-crl(XI), pro(~2(X1), pro-cr2(V), pro-cul(II), and pro-cu2(1) chains. Of the
14 cell lines analyzed, only 3 gave a positive signal whenhybridized to the COLllAl probe (Fig. 5). To be precise, two6426

Structure

COLI IAl

COL llA2

7.3+

-

6.3*

12

6.0*
4.8*

I234

34

COL IA2

COL 5A2

6.3*
5.0

and Expression of Type XI Collagen

*

I.-+

5.0*
4.5+

pro-Lul(X1) collagen chains are produced by these tumor lines
and, if so, to characterize into which parental trimers they
are assembled. Relevant to this, early studies have suggested
that the collagen of A-204 cultures consists solely of procrl(V) and pro-cyB(V) chains that undergo limited processing,in that medium collagen appears to retain large nontriple
helical domains (23). Such a finding should now be reconsidered in light of the results presented here, and of recent
structural data that are consistent with complete proteolytic
processing of the a2(V) propeptides (19, 24).
Acknowledgments-We
are very much indebted to Drs. B. Olsenand M. Young for providing us with the pro+2(XI) probe and bonederived cell lines, respectively, and to Dr. M. van der Rest for many
helpful suggestions and discussions. We thank R. Lingeza for the
preparation and typing of the manuscript.

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Res.
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87,1124-1131
I2
34
I2
34
2. Morris, N. P., and Bachinger, H. P. (1987) J. Biol. Chem. 262,
11345-11350
FIG. 5. Pattern of collagen expression.
In each of the four3.
Bernard,M., Yoshioka, H., Rodriguez, E., van der Rest, M.,
autoradiograms, the RNAs are from GM00637D (lane 1), A-204 (lane
Kimura, T., Ninomiya, H., Olsen, B. R., and Ramirez, F. (1988)2), HT-1080 (lane 3), and RD (lane 4). Sizes of mRNAs, in kilobases,
J. Biol. Chem. 263,17159-17166
are indicated on the left side of the panels. Each autoradiogram was
4. Henry, I., Bernheim, Bernard, M., van der Rest, M., Kimura, T.,
exposed for an identical period of time (48 h); each lane contains the
Jeanpierre, C., Barichard, F., Berger, R., Olsen, B. R., Ramirez,
same amount of poly(A)’ RNA (1 rg); each probe was used at theF., and Junien, C. (1988) Genomics 3,87-90
same concentration (5 rig/ml) and at a nearly identical specific
5. Kimura, T., Chea, K. S. E., Chan, S. D. H., Lui, V. C. H., Mattei,
activity (3 X 10’ cpm/rg). Note that the 6.3-kilobase pro-cr2(XI)
M. G., van der Rest, M., Ono, K., Solomon, E., Ninomiya, Y.,
mRNA species (5) cannot be seen in this particular exposure. Furand Olsen, B. R. (1989) J. Biol. Chem. 264,13910-13916
thermore, the COLlA2 band seen with RD RNA is a cross-hybridiBurgeson, R. E., Hedba, P. A., Morris, N. P., and Hollister, D.
zation product because it does not correspond to the size of a proW. (1982) J. Biol. Chem. 257, 7852-7856(u2(1) mRNA (22).
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and Functionofamounts of pro-cu2(1) and pro-cuB(X1) mRNAs (Fig. 5). In
Collagen Types (Mayne, R., and Burgeson, R. E., eds) pp. 261contrast, in the SV40-transformed fibroblast line GM0036D,
284, Academic Press, Orlando, FL
pro-aI(
pro-aB(XI), and pro-a2(V) mRNAs were present 10. Niyibizi, C., and Eyre, D. R. (1989) FEBS Lett. 242, 314-318
at levels significantly lower than pro-a2(1) (Fig. 5). This result 11. Weil, D., Bernard, M., Combates, N., Wirtz, M. K., Hollister, D.
W., Steinmann, B., and Ramirez, F. (1988) J. Biol. Chem. 263,
is consistent with the relative representation of each of these
8561-8564
collagen sequences in the GM0036D cDNA library (3,21,22).
12. Hattori, M., and Sakaki, Y. (1986) Anal. Biochem.
152,232-238
Relative amounts of pro-cu2(V) and pro-oll(X1) mRNAs were 13. Mount, D. W., and Conrad, B. (1987) Nucleic Acids Res. 14, llalso noted to be distinctly different in each of the two rhab16
domyosarcomas (Fig. 5). Type XI mRNAs were not expressed 14. Chirwing, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,
W. J. (1979) Riochemistryl&5294-5299
to a significant amount in other randomly chosen tumor lines,
15.
Thomas.
P.
S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77. 5201including an embryonal rhabdomyosarcoma, as well as in fetal
5205
or adult bone tissue, and amnion fibroblasts. Finally, in none 16. Perlman, D., and Halvorson, H. 0. (1983) J. Mol. Biol. 167,391of the cell lines analyzed in this manner were detectable levels
409
17. Timpl, R., and Glanville, R. W. (1981) Clin. Orthop. Relat. Res.
of pro-cul(I1) mRNA (data not shown).
158,224-242
In conclusion, the results are consistent with the previous
18. Su, M. W., Benson-Chanda, V., Vissing, H., and Ramirez, F.
suggestion (3) that COLllAl transcription occurs in tissues(1989) Genomics 4,438-441
other than cartilage, such as placenta, and some tumor or 19. Woodbury, D., Benson-Chanda, V., and Ramirez, F. (1989) J.
*

virally transformed cell lines. It could be argued that ectopic
gene expression in transformed cells is not physiologicallysignificant. It is, however, of some interest to note that theindependently co-expressed type XI gene, COLllA2, and the
major cartilage collagen gene, type II, do not display the same
pattern of expression as COLllAl. Hence, the two rhabdomyosarcomas may represent suitable experimental models for
elucidating the molecular factors involved in the selective
activation of this cartilage-specific collagen gene.
The data also provide the basis for an analysis of whether

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