Matrix Biology 24 (2005) 283 – 294
www.elsevier.com/locate/matbio

Pro-a3(V) collagen chain is expressed in bone and its basic
N-terminal peptide adheres to osteosarcoma cells
Kenji Yamaguchia, Noritaka Matsuoa, Hideaki Sumiyoshia, Noritaka Fujimotoa,
Ken-ich Iyamac, Shigetaka Yanagisawab, Hidekatsu Yoshiokaa,*aDepartment of Anatomy, Biology and Medicine, Oita University, Faculty of Medicine, 1-1 Hasama-machi, Oita 879-5593, Japanb
Department of Oncological Science, Faculty of Medicine, Oita University, 1-1 Hasama-machi, Oita 879-5593, Japanc
Department of Surgical Pathology, Kumamoto University School of Medicine, Kumamoto 860-8556, Japan
Received 11 August 2004; received in revised form 9 March 2005; accepted 9 March 2005

Abstract
The third a-chain of type V collagen, a3(V) chain, was initially identified in the placenta more than 20 years ago, but was poorly
characterized with regard to its expression and function. We generated a specific monoclonal antibody against the N-terminal domain of the
pro-a3(V) chain and examined gene expression using immunohistochemical methods combined with in situ hybridization. The pro-a3(V)chain was seen in funis and amnion, but not chorionic villi and deciduas of mouse placenta. In mouse embryo, the transcripts of the proa3(V) gene were seen in tissues that were related to bone formation as well as developing muscle and nascent ligament previously reported(J. Biol. Chem. 275, 8749 – 8759, 2000). However, immunohistochemistry showed that pro-a3(V) protein accumulated rather in thedeveloping bone of mouse embryo. On the other hand, the N-terminal globular domain of the pro-a3(V) chain has a unique structure that
contains a highly basic segment of 23 amino acids. The peptide derived from the basic segment showed a specific adhesive feature toosteosarcoma cells but not to chondrosarcoma cells. The four heparin binding sites in the basic segment equally contribute toward adhesion tothe osteosarcoma cells. Our data suggested that N-terminal globular domain of the pro-a3(V) chain influence bone formation of osteoblasts
through proteoglycan on the cell surface during development or regeneration.
D 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.
Keywords: Pro-a3(V) collagen; Bone formation; Heparin binding; Gene expression

1. Introduction
The collagens are major constituents of the extracellular matrix. Among them, fibrillar collagen, which
include five different molecular types, I, II, III, V and
XI, participate in the formation of fibrils (Vuorio and de
Crombrugghe, 1990; van der Rest and Garrone, 1991;
Brown and Timpl, 1995). Collagen V is a minor

Abbreviations: PBS, Phosphate-buffered saline; RT-PCR, Reverse
transcription-polymerase chain reaction; IPTG, Isopropyl-h-d-thiogalactopyranoside; GST, Glutathione S-transferase; ELISA, Enzyme-linked
immunosorbent assay; DMEM, Dulbecco’s modified Eagle’s medium.
* Corresponding author. Tel.: +81 97 586 5670; fax: +81 97 549 6302.
E-mail address: hidey@med.oita-u.ac.jp (H. Yoshioka).

component of connective tissues but it plays an importantrole in matrix organization. Mutations in the collagen V
gene produce abnormal fibril aggregates in the tissue(Andrikopoulos et al., 1995; Toriello et al., 1996; De
Paepe et al., 1997; Michalickova et al., 1998). Type V
collagen co-polymerizes with the major collagen, type I,
and regulates the diameter of collagen fibers in noncartilage tissues. Collagen V is widely distributed invertebrate tissues as an [a1(V)]2a2(V) heterotrimer(Fessler and Fessler, 1987; Fichard et al., 1994). Other
forms of collagen V include an [a1(V)]3 homotrimer
secreted by a line of Chinese hamster cells (Haralson etal., 1980) that may also exist in normal tissues (MoradiAméli et al., 1994; Kumamoto and Fessler, 1980), and apoorly characterized a1(V)a2(V)a3(V) heterotrimer that0945-053X/$ - see front matter D 2005 Elsevier B.V./International Society of Matrix Biology. All rights reserved.
doi:10.1016/j.matbio.2005.03.006

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K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

is primarily isolated from placenta (Rhodes and Miller,
1981; Niyibizi et al., 1984) but also reported in uterus,
skin, and synovial membranes (Fessler and Fessler, 1987;
Abedin et al., 1982).
Collagen specifically interacts with other macromolecules in the extracellular matrix such as fibronectin, laminin
and proteoglycan (Hynes and Yamada, 1982; Timpl and
Brown, 1994; Scott, 1988) and with cell-surface receptor,
integrins (Kramer and Marks, 1989; Plow et al., 2000;
Ruggiero et al., 1996). The interactions are important inregulating cell behavior, including proliferation, migration,and differentiation during development and physiologicaland pathological conditions. Heparin is abundant in thetissues as a form of heparin sulfate proteoglycans. Heparinbinding sites are found in the extracellular matrix proteinssuch as collagens, fibronectin, tenascin, and laminin.
Common structural motifs have been proposed from theanalysis of different heparin binding sites (Cardin andWeintraub, 1989).
Type V collagen, [a1(V)]2a2(V) form, was shown topossess a site that binds heparin/heparan sulfate underphysiological conditions (Cardin and Weintraub, 1989;
LeBaron et al., 1989). This site is located within the
NH2-terminal half of the a1(V) chain (Yaoi et al., 1990;
Delacoux et al., 1998). Delacoux et al. (2000) narrowed
the region down to a 12 kDa fragment that contains acluster of seven basic amino acids. Unlike a1(V) chains,
a2(V) and a3(V) chains do not bind heparin underphysiological or denaturing conditions (Mizuno andHayashi, 1996). Triple helical type V collagen trimers
bind to heparin with decreasing affinity in the order[a1(V)]3 > [a1(V)]2a2(V) > a1(V)a2(V)a3(V), indicatingthat a1(V) chains mediate heparin binding, but a2(V)and a3(V) chains do not (Mizuno and Hayashi, 1996).
Mann (1992) presented the first partial human amino acidsequence of the N-terminus of a3(V) collagen chain.
Recently, Imamura et al. (2000) provided the primarystructure of the human and mouse pro-a3(V) collagenchains. The structure of the pro-a3(V) chain was shown to
be closely related to that of the pro-a1(V) chain. It has aunique acidic domain in the N-terminal globular domain,which is also contained in the pro-a1(V) chain as well as
pro-a1(XI) and pro-a2(XI) chains. They showed expressionof the pro-a3(V) gene in the epimysial sheaths of developing
muscles, within nascent ligaments adjacent to forming bones
and in joints using in situ hybridization. Alternatively,
Chernousov et al. (2000) reported that rat pro-a4(V) chain
must be the counterpart of mouse and human pro-a3(V)chains based on its high similarity of structure as shown in
the genomic databases (Gopalakrishnan et al., 2004). Theirgroup also found a new heparan sulfate binding site thatmediates Schwann cell adhesion in the unique N-terminal
domain of rat pro-a4(V) (Erdman et al., 2002). They showedthat the major binding proteins are glypican-1 and perlecan
using pull down assays and immunofluorescent staining(Rothblum et al., 2004).

In this study, we generated a specific monoclonal
antibody directed against the unique N-terminal domain of
the pro-a3(V) chain. We examined the expression of the
pro-a3(V) chain using immunohistochemical methods andin situ hybridization. The pro-a3(V) chain was observed intissues related to bone formation. Furthermore, the peptide
derived from the basic segment flanking the acidic domainof the pro-a3(V) chain binds to osteosarcoma cells.

2. Results and discussion
2.1. Transcripts of pro-a3(V) collagen gene
The pro-a3(V) chain was originally isolated from humanplacenta but is distributed in many more tissues thanpreviously expected (Rhodes and Miller, 1981; Abedin etal., 1982; Imamura et al., 2000). Initially, to examine the
pro-a3(V) gene expression temporally and spatially inmouse, we performed RT-PCR. As shown in Fig. 1A, proa3(V) transcripts were detectable from E16.5, whereas proa1(V) transcripts were readily detectable in embryos at
E12.5. In different tissues of E18.5, relatively high levels of
pro-a3(V) expression were seen in placenta, kidney,vertebrae, calvaria and tongue (Fig. 1B). Weak bands werealso detectable in tail and heart, but almost undetectable inliver, lung, intestine, limb, brain and skin (Table 1). By
contrast, pro-a1(V) gene was expressed in many tissues
tested (data not shown) (Wu et al., 1998).
To characterize the expression pattern of the pro-a3(V)gene in detail, we applied in situ hybridization. In situ
hybridization was performed on sagittal sections of E16.5
mouse embryos using specific probes for pro-a3(V) basic/
acidic domain, pro-a1(V) acidic domain and type II
collagen (Yoshioka et al., 1995). Although pro-a1(V)Fig. 1. RT-PCR analysis of pro-a3(V) collagen gene expression in mouseembryo. A: RNAs from whole mouse embryos at E12.5 – E18.5 were used.
RT-PCRs using specific primers for the mouse pro-a3(V), pro-a1(V)collagen and GAPDH genes were performed. B: RNAs from different
tissues of the E18.5 mouse embryo were used. GAPDH was used as aninternal control. Expected size of the PCR product is shown on the right ofeach panel.

K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

285

Table 1
Expression of a3(V) collagen gene in the placenta and the mouse embryo

Placenta
Chorionic villi
Funis
AmnionDeciduas
Liver
Lung
Intestine
KidneyRenal facia
Tail
Vertebrae
Heart
VesselLimb
Calvaria
TongueBrain
Skin
Superficial facia
of developing
muscleRT-PCRIn situhybridization

Immunohistochemistry

+
nd
nd
nd
nd

/

/

+
+

+
+

+

+

+

+

+
+

+
+

+

nd

+

+

+
nd
T
+
T
nd

+, Positive; T, weakly positive; , negative; nd, not determined.
The E18.5 mouse embryos were used for RT-PCR, and E16.5 for in situ
hybridization and immunohistochemistry.

expression was widely distributed throughout developing
connective tissues (Fig. 2C), pro-a3(V) expression wasrestricted (Fig. 2B). Type II collagen was strongly expressedin cartilage, especially prehyper/hypertrophic chondrocytes(Figs. 2D and 3H). Pro-a3(V) mRNA was expressed inosteoblasts of calvaria (Fig. 3C) and in the associatedperiosteal cells surrounding first ossification center ofvertebrae (Fig. 3D). The former represent bones formed
by intramembranous ossification, and the latter by endochondrial ossification. By contrast, pro-a1(V) expression
was seen in calvaria and its surrounding connective tissue(Fig. 3E), and type II collagen was never seen in calvaria(Fig. 3G). In vertebrae, pro-a1(V) mRNA was expressed inperichondrial cell layers surrounding cartilage primordium(Fig. 3F).

Fig. 3. In situ hybridization of the tissues related to the bone formation of
E16.5 mouse embryos. Photomicrographs are H – E stained with brightfield(A and B) or hybridization with darkfield (C – H). Panels are shown at highmagnification of Fig. 2 to clearly show the portion of calvaria (A, C, E and G)
and vertebrae (B, D, F and H). The sections were hybridized with aradioactively labeled pro-a3(V) (C and D), pro-a1(V) (E and F) and a1(II)(G and H) collagen antisense riboprobe. Specific regions were labeled asfollows: skin (Sk), subcutaneous connective tissue (CT), calvaria (frontalbone) (Ca), midbrain (Br), first ossification center (OsC), hypertrophic
chondrocyte (Hc), prehypertrophic chondrocyte (Pc), ganglion (Ga). Scalebar: 100 Am.

2.2. Distribution of pro-a3(V) collagen chain in bonerelated tissues
To detect the protein in tissues by immunohistochemistry, we prepared a specific monoclonal antibody directedagainst the basic/acidic domain of pro-a3(V). The antibody

Fig. 2. In situ hybridization of E16.5 mouse embryos. Photomicrographs are hematoxylin and eosin (H – E) staining with brightfield (A) or in situ hybridization
with darkfield (B – D). Sagittal sections from E16.5 embryos were hybridized with a radioactively labeled pro-a3(V) (B), pro-a1(V) (C) and a1(II) (D) collagen
antisense riboprobe. Scale bar: 1 mm.

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K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

A

a

B

1

2

3

b

4

c

d

C
1.2
α3(V)-GST

1.0

200K

α3(V)-His
α1(V)-GST

O.D.

0.8
0.6
0.4
0.2

118K

0.0
x20

x40

x80

x160
dilution

x320

x640

x1280

Fig. 4. Characterization of monoclonal antibody against the basic/acidic domain of pro-a3(V) chain. A: Western blot analysis using recombinant proteins.
Purified recombinant proteins were run on a 15% SDS-PAGE gel and stained with Coomassie brilliant blue (panel a). The proteins blotted on the filters were
hybridized with anti-pro-a3(V) antibody (panel b), anti-pro-a1(V) antibody (panel c) and anti-GST antibody (panel d). The samples in each lane were as
follows—lane 1: pro-a3(V) – GST fusion protein, lane 2: pro-a1(V) – GST fusion protein, lane 3: empty vector (GST), lane 4: pro-a3(V) – His-tag fusion protein,lane M: marker. The positions of the molecular mass standards are shown on the left. B: Western blot analysis using native collagenous protein. Proteins were run
on a 10% SDS-PAGE gel and stained with Coomassie brilliant blue (lanes 1 and 2). The proteins blotted on the filters were hybridized with anti-pro-a3(V)antibody (lanes 3 and 4). The samples were digested with collagenase (lanes 2 and 4). The positions of the molecular mass standards are shown on the left. C:
ELISA using anti-pro-a3(V) antibody. The pro-a3(V) monoclonal antibody reacted with the pro-a3(V) – GST (open circles) and pro-a3(V) – His (closed
squares) recombinant protein applied to a plate as a serial dilution, but not with similar dilutions of the pro-a1(V) – GST (closed triangles) fusion proteins.

was purified by protein G affinity column chromatography and Western blot and ELISA confirmed its specificity. As shown in Fig. 4A and C, the anti-pro-a3(V)antibody recognized the GST fusion and His-tagged proa3(V) recombinant polypeptide but not the tagged proa1(V) recombinant polypeptide. The antibody also recognized a collagenous peptide of approximately 220 kDa
from a fraction of neutral salt-extracted osteosarcoma cells(Fig. 4B). An anti-pro-a1(V) polyclonal antibody directedagainst the acidic domain was also generated.
Sagittal sections from E16.5 mouse embryos, as used in
the in situ hybridization experiments, were analyzed byimmunohistochemistry. At low magnification, the pro-a3(V) chain was observed in tissues that are related tobone formation, such as the calvaria, vertebra, maxilla,mandibula, and clavicula (Fig. 5C). At this developmental
stage, alkaline phosphatase activity was detected strongly
in the frontal region of the calvaria, perichondrium, andbone matrix (Fig. 5B). Both staining patterns were
essentially consistent. Pro-a1(V) collagen was detected inmost connective tissues, including bone-related tissues(Fig. 5D). In the calvaria region, strong expression of
pro-a3(V) was observed at high magnification, in the matrixof the calvaria and aponeurosis (Fig. 6E), whereas pro-a1(V)was observed in the matrix of the calvaria and subcutaneous
tissue, but not in the aponeurosis (Fig. 6G). In the vertebral

Fig. 5. Immunohistochemical localization of pro-a3(V) chain in E16.5 mouse embryos. Photomicrographs were shown for sagittal serial sections of an E16.5
mouse embryo. Paraffin sections were stained with H – E (A), alkaline phosphatase activity (B), anti-pro-a3(V) antibody (C) and anti-pro-a1(V) antibody (D).
Scale bar: 1 mm.

K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

287

jelly, in funis and amnion (Fig. 7B and E). However, they
could never be seen in chorionic villi and decidua. The
immunohistochemical data were consistent with those of insitu hybridization (Fig. 7C and F).
Imamura et al. (2000) reported that the pro-a3(V) gene
was expressed in the epimysial sheaths of developing
muscles and within nascent ligaments adjacent to formingbones and joints in the E15.5 mouse embryo. Similarly, we
could detect the signals in those regions in the E16.5 mouseembryo. The staining of pro-a3(V) could be detected in the
superficial fascia of developing muscle and renal fascia
around the kidney and adrenal gland (Fig. 7I and L).
However, we could not clearly detect a signal in otherregions where expression was positive by RT-PCR or in situ
hybridization (Table 1). This discrepancy may be due to thefast turnover or disseminated distribution of the protein inthese tissues.
2.4. Cell adhesion of pro-a3(V) chain

Fig. 6. Immunohistochemical localization of pro-a3(V) chain in the tissues
that are related to bone formation in E16.5 mouse embryos. Photomicrographs are H – E stained (A and B) or immunohistochemical stained (C – H).
Panels are shown at a higher magnification than Fig. 5 to clearly show theportion of calvaria (A, C, E and G) and vertebrae (B, D, F and H). The
sections were stained for alkaline phosphatase activity (C and D), anti-proa3(V) (E and F) and anti-pro-a1(V) (G and H) antibodies. Specific regions
were labeled as follows: skin (Sk), subcutaneous connective tissue (CT),calvaria (frontal bone)(Ca), midbrain (Br), aponeurosis (Ap), first ossification center (OsC), hypertrophic chondrocyte (Hc), prehypertrophic chondrocyte (Pc), perichondrial (Pe), periosteal (Po). Scale bar: 100 Am.

region, pro-a3(V) was detected in the periosteum and bonematrix, where ossification initially occurs (Fig. 6F). This
staining pattern is consistent with that of alkaline phosphatase (Fig. 6D). Weak staining of the pro-a3(V) chain
was also seen in ligamentous attachments. On the other
hand, staining for pro-a1(V) was more intense in the
periosteum and ligamentous attachments than in the bonematrix (Fig. 6H). This pattern differed from that of alkaline
phosphatase.
2.3. Expression in non-bone-related tissues
The a3(V) chain was originally isolated from theplacenta. As shown in Fig. 1B, the transcripts of proa3(V) were detected in the placenta using RT-PCR.
However, Chernousov et al. (2000) did not detect proa3(V) in the placenta of rat. This was one of the reasons forthem to call the collagenous peptide, the a4(V) chain, notthe a3(V) chain. We examined the distribution of the mousepro-a3(V) chain in the placenta using in situ hybridizationand immunohistochemical methods. Signal from the transcripts were seen in the soft connective tissue, Wharton’s

To elucidate the biological function of type V collagen in
osteoblast, we used an osteosarcoma cell line to examine
whether the N-terminal domain of pro-a3(V) chain is
involved in cell adhesion. The N-terminal globular domainof the pro-a3(V) chain can be divided into subdomains(Fig. 8). Among them, the acidic domain of pro-a3(V)chain, which is flanked by a highly basic segment of 23
amino acids at the N-terminus, is unique because it is only
found in mouse and human pro-a3(V) and rat pro-a4(V)chains (Imamura et al., 2000; Chernousov et al., 2000).
We tested the adhesive ability of the basic/acidic domainof pro-a3(V) chain using recombinant proteins to ROS andRCS cells. As shown in Fig. 9, ROS cells adhere to the basic/
acidic domain of pro-a3(V) recombinant protein but not tothe acidic domain of pro-a1(V). The adhesion of RCS cells
was hardly recognizable in both acidic domains of pro-a1(V)and basic/acidic domain of pro-a3(V). This adhesivefunction was restricted to the region of 239 –269 aminoacids where the basic segment is contained. This segment has
four repeats of the heparin binding consensus sequence
BBXB (B and X are a basic and any amino acids,
respectively) (Fig. 12A) (Cardin and Weintraub, 1989). To
assess the heparin binding activity of the recombinant
protein, heparin affinity chromatography was performed.
As shown in Fig. 10A and B, pro-a3(V) (239 –369) and proa3(V) (239 – 269) fragments, containing the basic segment,bound to the heparin column and were eluted at a high NaCl
concentration, reflecting high affinity binding to heparin. In
contrast, the pro-a1(V) acidic domain and pro-a3(V)(269 –
369) that deletes the basic segment, dramatically abolishedthe heparin binding activity. Thus, the basic segment in the
N-globular domain of pro-a3(V) chain is important to bindheparin.
These results suggest that ROS cells adhesion to proa3(V) peptide might be mediated by cell-surface heparansulfate proteoglycans. The contribution of glycosaminogly-

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K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

Fig. 7. Expression in non-bone-related tissues. Photomicrographs are H – E stained (A, D, G and J), in situ hybridization with darkfield (B, E, H and K) andimmunohistochemical staining (C, F, I and L). The sections were hybridized with a radioactively labeled pro-a3(V) collagen antisense riboprobe (B, E, H andK) and stained for anti-pro-a3(V) collagen antibody (C, F, I and L). The tissues are placenta (A – C), funis attached to fetal abdomen (D – F), superficial fascia
of developing muscle (G – H) and kidney and adrenal gland (J – L). Specific regions were labeled as follows: chorionic villi in placenta (Pl), amnion (Am), funis(Fu), superficial fascia in fetus (Fa), skin (Sk), vertebrae (Ve). Scale bar: 100 Am.

can to ROS adhesion was examined by plating the cells onto
the recombinant proteins of pro-a3(V) basic/acidic domain
in the presence of heparin, chondrotin sulfate and dextran

sulfate. As shown in Fig. 11, heparin and dextran sulfate,added at a concentration of 10 Ag/mL, inhibited celladhesion by 76%. Chondrotin sulfate inhibited adhesion

α1(V)
(37)

(217)

(189)

(115)

(1014)(266)

S
NH2 - P

Npp

AD

SC/NT

COL

Cpp

255

443

-COOH

α1(V) 255/443

α 3(V)(29) (209)S
NH2 - PNpp

(70)(1011)(251)

BS/AD SC/NT

(133)

COL

Cpp

371

239

239

269

270

-COOH

α 3(V) 239/371
α 3(V) 239/269371

α 3(V) 270/371

Fig. 8. The constructs of recombinant protein of the mouse pro-a3(V) and pro-a1(V) collagen polypeptides. The recombinant protein covered the basic/acidic
domain of pro-a3(V) and the acidic domain pro-a1(V) chains were generated. The domains of prepro-a3(V) and prepro-a1(V) chain are consist of a signal
peptide (SP), N-terminal propeptide (Npp), basic/acidic (BS/AD) or acidic domain (AD), short collagenous segment (SC), N-telopeptide (NT), central
continuous collagenous domain (COL) and C-terminal propeptide (Cpp). A short bar in the pro-a3(V) chain indicates a basic segment flunked acidic domain.
Vertical arrows and numbers in parenthesis indicate the cleaved sites of pro-a1(V) chains and number of amino acid residues in individual domains,respectively. The recombinant protein numbers indicate the number of amino acid residues from the N-terminal end.

cell adhesion (absorbance 595nm)

K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

289

1
0.9
0.8

ROS
RCS

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

GS

GS

GS

GS

GS

T

T

T

T

T

α

α

α

α

3(

3(

3(

1(

69

43

71

9/2

9/3

5/4

71
0/3
27
V)

23
V)

23
v)

25
V)

Fig. 9. Cell adhesion to the recombinant proteins of pro-a3(V) and pro-a1(V) collagen polypeptides. ROS and RCS cells were plated on dishes coated with therecombinant proteins. Cell adhesion was quantitated by staining with crystal violet.

sites, we replaced each heparin binding site by sitedirected mutagenesis. These amino acids were replaced byalanine (Fig. 12A). The mutated fusion proteins wereexpressed in bacteria and tested by the heparin binding
assay and cell adhesion assay. As shown in Fig. 12B, M1,
M2, M3 and M4 mutated proteins were eluted from theheparin column at NaCl concentration of 0.8 – 1.5 M.
However, the binding affinities were decreased comparedwith that of wild type pro-a3(V)(247 – 269), which eluted
at the concentration of 1.0 –2.0 M. Similarly, as shown in
Fig. 12C, each mutated protein, M1, M2, M3 and M4,
showed approximately 60– 70% of the adhesive activity to
ROS compared with that of wild type pro-a3(V)(247 –
269). These results indicate that each heparin binding siteby about 30%. The interaction between ROS cells and basic
segment might be mediated by a cell-surface heparan sulfate
proteoglycan receptor. Erdman et al. (2002) showed the Nterminal binding activity to Schwann cells. Rothblum et al.
(2004) suggested that this binding is mediated via glypican1, that is a lipid-anchored proteoglycan of the plasma
membrane. In osteoblasts, the same or similar proteoglycan
might also contribute to the interaction with the basic
segment.
2.5. Adhesion activity of the four heparin binding sites
To know which heparin binding site is the mostimportant in cell adhesion among the four heparin bindingA
absorbance (280nm)

0.08
0.07

GST α1(V)255/443
α

0.06
0.05

GST α3(V)239/371α
GST α3(V)239/269
α

0.04

GST α3(V)270/371
α

0.03
0.02
0.01
0
0.2

0.4

0.6

0.8

1.0

1.25

1.5

2.0

NaCl (M)

B

F

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.25

1.5

2.0 (M)

GST α
α1(V)255/443
GST α3(V)239/371αα3(V)239/269
GST αα3(V)270/371
GST α

Fig. 10. Heparin binding of the recombinant pro-a3(V) and pro-a1(V) collagen polypeptides. GST fusion proteins of pro-a3(V) and pro-a1(V) collagen
polypeptides were applied to a heparin – sepharose column and eluted by step gradient at the indicated concentrations from 0 to 2.0 M NaCl. A: Elution data areshown in the graph. B: Aliquots of column fractions were subjected to Western blot analysis with anti-GST antibody. F indicates the fraction of flowthrough.

K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294
1.2

ize its function in bone formation during development andregeneration.

1.0
0.8
0.6

3. Materials and methods

0.4

3.1. Animals

0.0

on
ch

x
de

p
he

(-)

Fig. 11. Inhibition of cell adhesion by glycosaminoglycan. ROS cells weresuspended in serum-free medium containing 10 Ag/mL of heparin (hep),
dextran sulfate (dex), and chondroitin sulfate (chon), and plated onto wells
coated with pro-a3(V)(239 – 369) recombinant protein. The values shown
are the mean T SD of five independent wells.

in the pro-a3(V) chain equally contributes with regard toadhesive interactions with ROS cells.
Delacoux et al. (2000) showed that a heparin binding site
is present in the collagenous domain of the a1(V) chains.
The sequence contains a cluster of seven basic amino acids
that are different from the consensus sequence of the heparinbinding site previously reported (Cardin and Weintraub,1989). However, its binding affinity is significantly lower
than that of the pro-a3(V) chain. The former is eluted at
0.35 M NaCl concentration whereas the latter is at 1.0 – 2.0
M. This basic segment would play a specific role through astrong electrostatic interaction with molecules such as
heparan sulfate proteoglycan in vivo. This portion of theprotein is retained in the fibrils that contain the rat a4(V)chain synthesized by Schwann cells (Chernousov et al.,
1996; Chernousov et al., 2000) and is exposed on the fibril
surface (Linsenmayer et al., 1993). However, Gopalakrishnan et al. (2004) have recently reported that this portion of
the recombinant human pro-a3(V) chain was removed by
BMP-1 in a fibroblast culture system. The processing of the
N-terminal domain of the pro-a3(V) chain (or rat a4(V)chain) may depend on the cell type or the circumstances of
the tissue. The processing of the pro-a3(V) chain in bone is
unknown. Whether the basic segment is retained in the
fibrils or removed, it might still affect the osteoblast in thedeveloping bone.
2.6. Conclusion
Using immunohistochemistry and in situ hybridization,
this study has demonstrated that the pro-a3(V) chain isexpressed in tissues that are related to bone formation as
well as in the funis and amnion of the placenta. The
peptide derived from the basic segment of the N-terminal
domain of the pro-a3(V) chain showed specific adherence
to osteosarcoma cells but not to chondrosarcoma cells.
The unique segment should play an important role in boneformation. Further study is required to determine theprecise role of this chain in osteoblasts, and to character-

Mice and rabbits were purchased from commercial
sources (Yoshitomi, Fukuoka, Japan; Kyudo, Saga, Japan).

Abasic segment
247

sequence269

PPRRKGKGKKKGRGRKGKGRKKNKETSEL

V)247/269WT
α3(V)247/269WT-----------------------

M1---AAAA----------------------

M2--------AAAA-----------

M3-------------AAAA------

M4-------------------AAAA---B

Absorbance (280 nm)

0.2

WT
M1
M2
M3
M4

0.08
0.06
0.04
0.02
0

0.1

0.5

1.0
NaCl (M)

1.5

*

*

M2

M3

2.0

C
cell adhesion (absorbance 595nm)

cell adhesion (absorbance 595nm)

290

1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

*

WT

M1

*

M4

Fig. 12. Assessment of four heparin binding sites in the basic segment of
pro-a3(V) chain for heparin binding affinity and ability of cell adhesion. A:
Amino acid sequence of the constructs with an alanine mutation. Note that
it was difficult to generate a 23 amino acids-size pro-a3(V)M1 and proa3(V)M4 using the PCR procedure because of annealing problems with theprimers. Therefore, pro-a3(V)M1 and pro-a3(V)M4 are six and threeamino acids, respectively, longer than the others. B: Heparin binding assay
using substituted mutant proteins. Bound proteins were eluted by step
gradient of indicated concentrations of 0 – 2.0 M NaCl. Elution data areshown in the graph. C: Cell adhesion assay using substituted mutant
proteins. ROS cells were plated on dishes coated with mutated protein. The
results are displayed graphically. The values shown are the mean T SD for
four independent wells. *p < 0.01.

K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

The animals were treated in accordance with the Oita
University Guidelines for the Care and Use of Laboratory
Animals based on the National Institutes of Health Guide
for the Care and Use of Laboratory Animals.
3.2. Staging of mouse embryos and preparation of sections
Gestational age was initially determined by the date offormation of the copulation plug and confirmed by crownrump length. For in situ hybridization and immunohistochemistry, mouse embryos were fixed overnight in fresh 4%
paraformaldehyde in phosphate-buffered saline (PBS),
dehydrated, and embedded in paraffin, and 7 Am consecutive sections were prepared.
3.3. Reverse transcription-polymerase chain reaction(RT-PCR)
RNA samples were prepared from E12.5, 14.5, 16.5, and18.5 mouse whole embryos, and from different tissues of theE18.5 mouse embryo, using Isogen (Wako, Osaka, Japan)
according to the manufacturer’s instructions. Total RNAs
were used as templates to synthesize cDNAs using Moloneymurine leukemia virus reverse transcriptase (Invitrogen,
Carlsbad, CA) and random hexamers. The PCR program
was as follows: 1 min at 94 -C for denaturation, 1 min at 55-C for annealing, and 1 min at 72 -C for extension; repeatedfor 30 cycles with a final extension of 7 min 30 s at 72 -C.
The gene-specific primers used in the PCR reactions were:
pro-a1(V)255/443: (forward) 5V-CAGGACCCTAACCCGGATGA-3V(reverse) 5V-CTCAAAGATGGTGTCCTGGT-3V
pro-a3(V)239/371: (forward) 5V-GATGAACCAGAAACCCCTGC-3V(reverse) 5V-AGCACCAGGAAAGATCTGGA-3V
GAPDH: (forward) 5V-AGAGGTGCTGCCCAGAACATCATC-3V(reverse) 5V-GTGGGGAGACAGAAGGGAACAGA-3V
3.4. In situ hybridization
In situ hybridizations were carried out using [35S]-labeledriboprobes on tissue sections of mouse embryos as
described previously (Iyama et al., 2001; Sumiyoshi et al.,2001). The fragments of the acidic domain of the pro-a1(V)and the basic/acidic domain of pro-a3(V) mouse collagen
cDNA were generated by the RT-PCR procedure mentioned
above. The amplified fragments were cloned into TA-Easy
vector (Promega, Madison, WI). Type II collagen cDNA
was described elsewhere (Yoshioka et al., 1995). After
linearization at appropriate restriction sites, sense andantisense probes were generated by in vitro transcription
with T3 and T7 polymerases. In situ hybridization was
performed on deparaffinized and proteinase K-treatedsections. The sections were incubated with [35S]-labeled291

antisense and sense cRNA riboprobe at 52 -C for 16 h andwashed several times with increasingly stringent conditions.
The slides were dipped in Kodak NTB-2 (Tokyo, Japan),dried for 1 h and exposed for 7 days at 4 -C. Sections werecounterstained with hematoxylin and eosin. Photographs
were taken by a camera attached to a microscope (Olympus
BX-50 and Keyence VB-6000/6010).
3.5. Expression of recombinant proteins
cDNAs encoding the different region of the basic/acidic
domain of the pro-a3(V) collagen (Fig. 7) were generated
by RT-PCR procedure using the pro-a3(V)239/371 fragment as a template.
Pro-a3(V)239/269:(forward) It is the same as the forward
primer of pro-a3(V)239/371.
(reverse) 5V-CTTGTTTTTCTTTCTTCCCT-3V
pro-a3(V)270/371: (forward) 5V-GAGACCTCAGAGCTGAGTCC-3V(reverse) It is the same as the reverse primer of proa3(V)239/371.
Various mutant cDNAs covering the basic segment of the
pro-a3(V) collagen were also generated using the proa3(V)239/269 fragment as a template.
Pro-a3(V)247/269WT: (forward) 5V-CGTCGTCGAAAGGGCAAAGG-3V(reverse) 5V-CTTGTTTTTCTTTCTTCCCT-3V
pro-a3(V)247/269M1: (forward) 5V-CCTCGTCGTGCAGCGGCCGCAGGGAAGAAA-3V(reverse) 5V-CAGCTCTGAGGTCTCCTTGT-3V
pro-a3(V)247/269M2: (forward) 5V-CGTCGTCGAAAGGGCAAAGGGAAGGCAGC, AGCGGCGGGT-3V(reverse) It is the same as the reverse primer of proa3(V)247/269WT.
pro-a3(V)247/269M3: (forward) It is the same as theforward primer of pro-a3(V)247/269WT.
(reverse) 5V-CTTGTTTTTCTTTCTTCCCGCGGCGGCTGCACC-3V
pro-a3(V)247/269M4: (forward) It is the same as theforward primer of pro-a3(V)247/269WT.
(reverse)5V-TGAGGTCTCCGCGGCTGCCGCTCTTCCCTT-3V
The amplified fragment was subcloned into the TA-Easy
vector (Promega). Following digestion at appropriate
restriction sites, the fragments were subcloned into pGEX4T vector (Amersham-Bioscience) to produce glutathione Stransferase (GST) fusion proteins. And His-tag pro-a3(V)recombinant protein was also prepared (Novergen, Darmstadt, Germany).
Nucleotide sequences of the inserts and junctions linked
to the vector were determined by automated DNA sequencing (ABI PRISM 310 Genetic Analyzer, Applied Biosys-

292

K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

tems, Foster City, CA) using the BigDye Terminator Cycle
Sequencing Kit (Applied Biosystems).
Large-scale preparation of bacterial sonicates for the
purification of GST fusion proteins were performed
according to the manufacturer’s protocol (AmershamBioscience). In brief, a single colony of E. coli BL21 cells
containing the recombinant pGEX plasmid was grownovernight and used to inoculate 2  YT medium containing
100 Ag/mL ampicillin and 2% glucose. The cells weregrown at 37 -C until OD600 of 0.5 was obtained, and protein
expression was induced by incubation for an additional 3
h in 0.1 mM isopropyl-h-d-thiogalactopyranoside (IPTG).
The cells were collected by centrifugation, resuspended in
PBS, sonicated, solubilized in 1  PBS and 1% Triton-X100, and bound to glutathione sepharose 4B. The glutathione fusion protein matrix was washed three times in fivebed volumes of 1  PBS and eluted with glutathione elution
buffer (10 mM reduced glutathione, 50 mM Tris – HCl pH
8.0). The purified proteins were collected and concentrated
by using Centricon (Millipore, Bedford, MA).
3.6. Production of anti-pro-a3(V) and anti-pro-a1(V)antiboies
Antibody against the pro-a3(V) basic/acidic domain wasestablished by the rat lymph node method (Kishiro et al.,1995). In brief, the purified GST recombinant protein was
mixed with an equal volume of Freund’s complete or
incomplete Adjuvant (Wako). Initial subcutaneous injections contained 100 Ag of recombinant protein withcomplete Freund’s adjuvant. Two booster injections, whichcontained the same amount of protein with incomplete
Freund’s adjuvant, were given 1 and 2 weeks after the initial
injection. The rats were bled 3 weeks after the second
booster injection. The inguinal lymph nodes were dissectedout for production of monoclonal antibody. Antigen against
the pro-a1(V) acidic domain was prepared by immunizing
rabbits using purified GST recombinant protein as described
previously (Iyama et al., 2001). Specificity of the antibodies
was confirmed by ELISA (enzyme-linked immunosorbent
assay) as previously described (Iyama et al., 2001) andWestern blot analysis (see below).
3.7. Cell culture
Rat ROS 17/2.8 osteosarcoma cells and rat RCS
chondrosarcoma cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% FBS,
penicillin (10 IU/mL), streptomycin (10 Ag/mL) andglutamine (200 Ag/mL). The cell lines were maintained at
37 -C in a humidified environment of 5% CO2.

medium described above plus 50 Ag/mL l-ascorbic acid and
50 Ag/mL h-aminopropionitrile to promote collagen synthesis and prevent cross-linking. Cells were lysed in
extraction buffer containing 50 mM Tris –HCl pH 7.5,
100 mM NaCl, 0.5% NP-40, 2.5 mM EDTA and 0.2 mM
PMSF. An aliquot of the sample was digested with bacterial
collagenase (Form III Wakojunyaku, Tokyo, Japan) before
loading onto the gel. The sample for digestion was firstneutralized with 0.5 M NaOH and then incubated withbacterial collagenase solution (250 U/mL collagenase, 50
mM Tris – HCl, pH 7.5, 0.2 M NaCl, 5 mM CaCl2)
containing 5.5 mM CaCl2 for 5 h at 37 -C.
Native collagenous and recombinant proteins were
electrophoresed on 10% and 15% polyacrylamide gels,
respectively. Proteins were transferred electrophoretically at
60 V for 5 h onto fluoro transmembrane (Japan Genetics,
Tokyo, Japan). The blotted membrane was treated with ablocking solution (PBS containing 4% nonfat milk) at 4 -C
overnight. The membrane was subsequently incubated atroom temperature for 1 h with the primary antibody. Afterbeing washed, the membrane was incubated with anti-rat or
rabbit-IgG-HRP at 1 : 5000 dilution (Wako) at room
temperature for 1 h and finally immunoreactive signals
were detected with ECL Plus Western Blotting Detection
Reagents (Amersham Biosciences).
3.9. Immunohistochemistry
For immunostaining of pro-a3(V) and pro-a1(V) collagen, deparaffinized sections were pretreated for antigen
retrieval by autoclave heating (121 -C, 110 kPa) in 10
mmol/L citrate buffer (pH 4.0) for 5 min (Iyama et al.,2001). These sections were blocked for endogenous
peroxidase activity with 1% H2O2 methanol for 30 min
and then washed in PBS. Subsequently, sections werepretreated for removal of glycosaminoglycan by 500 U/mL
hyaluronidase (Sigma) for 20 min at 37 -C. Thereafter,
sections were immersed in 5% normal rabbit serum in PBS
for 30 min, covered with anti-pro-a3(V) monoclonal or antipro-a1(V) polyclonal antibody, and incubated for 18 h at 4
-C. Immunoreactions were performed using a Vectastain
peroxidase ABC kit (Vector Laboratories, Burlingame, CA).
The antigenic sites were demonstrated by reacting thesections with a mixture of 0.05% 3,3V-diaminobenzidine
tetrahydrochloride (Dojin Chemicals, Tokyo, Japan) in 0.05
mol/L Tris – HCl pH 7.6, containing 0.01% H2O2 for 7 min.
After washing in distilled water, the nuclei were stained withmethylgreen, and then the sections were dehydrated inethanol, cleared in xylene, and mounted in Permount (Fisher
Scientific).
3.10. Heparin affinity chromatography

3.8. Western blot analysis
A fraction containing collagenous protein was preparedfrom the cell layer of ROS cells. Cells were cultured in

Purified recombinant protein was subjected to heparin
affinity chromatography on prepacked HiTrap-Heparin HP
columns (Amersham Biosciences). The columns were

K. Yamaguchi et al. / Matrix Biology 24 (2005) 283 – 294

equilibrated with 50 mM Tris –HCl pH 7.5 at a flow rate of
0.5 mL/min. The proteins were diluted in equilibrationbuffer, and 500 Ag of each protein was applied to thecolumn. The column was eluted at a flow rate of 0.5 mL/
min with 50 mM Tris – HCl pH 7.5 followed by a linear
gradient of 0 – 2.0 M NaCl in 50 mM Tris – HCl pH 7.5.
3.11. Cell adhesion assay
The recombinant proteins were used to coat 24-wellplates (2 Ag/cm2). The proteins were diluted in 20 mM
Tris –HCl pH 7.5, 100 mM NaCl to a final concentration of
10 Ag/mL, and incubated in the wells at 37 -C for 18 h. The
solution was removed, and the plates were blocked with asolution of 1% bovine serum albumin in 20 mM Tris – HCl
pH 7.5, 100 mM NaCl at 37 -C for 1 h. The blocking
solution was removed, and the wells were washed with
PBS. ROS and RCS cells were removed from the dishes bytrypsinization. ROS and RCS cells were harvested bycentrifugation, resuspended in serum-free medium, andadded to the protein-coated wells. The plates were incubatedfor 3 h at 37 -C. At the end of the incubation period, themedium was aspirated, and the wells were washed withDMEM to remove nonadherent cells. Attached cells were
fixed with 3% paraformaldehyde in PBS and stained with
0.5% crystal violet in 10% ethanol for 20 min. Afterextensive washing with water, bound dye was solubilizedwith 1% SDS, and the absorbance was read at a wavelengthof 595 nm.

Acknowledgements
We thank the staff of Division of Biomolecular Medicine
and Medical Imaging, and Division of Radioisotope
Research, Institute of Scientific Research, Oita University
where we performed some experiments. This work was
supported by Grants-In-Aid for Scientific Research(11470312 and 14370468 to H.Y.) from the Ministry of
Education, Culture, Sports, Science and Technology ofJapan, and Grant of Possible Trial (to H.Y.) from The Oita
Prefectural Organization For Industry Creation.

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