Degradation of vitronectin by matrix metalloproteinases-1, -2, -3, -7 and -9
ABSTRACT The susceptibility of vitronectin (Vn) purified from human plasma to digestion by matrix metalloproteinases (MMPs) was examined. MMP-2, -3, -7 and -9 except for MMP-1 degraded Vn into multiple fragments. MMP-7 showed the highest activity to the substrate among these MMPs, digesting 8-, 30- and 44-fold more preferentially than MMP-2, -3 and -9, respectively. These data suggest that MMP-2, -3, -7 and -9 may be responsible for the pathological degradation and/or normal turnover of Vn.
- [show abstract] [hide abstract]
ABSTRACT: Matrix metalloproteinases (MMPs) are important mediators of neural crest (NC) cell migration. Here, we examine the distribution of tissue inhibitor of metalloproteinase (TIMP) -2 and TIMP-3 and test whether manipulating TIMP levels alters chicken cardiac NC cell migration. TIMP-2 mRNA is expressed at stage 11 in the neural epithelium and only in migrating cardiac NC cells. TIMP-3 mRNA is expressed only in the notochord at stage 8 and later in the outflow tract myocardium. Exogenous TIMP-2 increases NC motility in vitro at low concentrations but has no effect when concentrations are increased. In vitro, NC cells express membrane type-1 matrix metalloproteinase (MT1-MMP) and TIMP-2 and they secrete and activate proMMP-2. Antisense TIMP-2 oligonucleotides block proMMP-2 activation, decrease NC cell migration from explants, and perturb NC morphogenesis in ovo. Because TIMP-2 is required for activation of proMMP-2 by MT1-MMP, this finding suggests TIMP-2 expression by cardiac NC cells initiates proMMP-2 activation important for their migration.Developmental Dynamics 01/2005; 231(4):709-19. · 2.59 Impact Factor
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ABSTRACT: Kawasaki disease (KD) is a complex disease, leading to the damage of multisystems. The pathogen that triggers this sophisticated disease is still unknown since it was first reported in 1967. To increase our knowledge on the effects of genes in KD, we extracted statistically significant genes so far associated with this mysterious illness from candidate gene studies and genome-wide association studies. These genes contributed to susceptibility to KD, coronary artery lesions, resistance to initial IVIG treatment, incomplete KD, and so on. Gene ontology category and pathways were analyzed for relationships among these statistically significant genes. These genes were represented in a variety of functional categories, including immune response, inflammatory response, and cellular calcium ion homeostasis. They were mainly enriched in the pathway of immune response. We further highlighted the compelling immune pathway of NF-AT signal and leukocyte interactions combined with another transcription factor NF- κ B in the pathogenesis of KD. STRING analysis, a network analysis focusing on protein interactions, validated close contact between these genes and implied the importance of this pathway. This data will contribute to understanding pathogenesis of KD.Computational and Mathematical Methods in Medicine 01/2013; 2013:989307. · 0.79 Impact Factor
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ABSTRACT: Decorin (DCN) is a ubiquitous proteoglycan comprised of a core protein attached to a single dermatan/chondroitin sulphate glycosaminoglycan chain. It may play a role in regulation of collagen fibrillogenesis and function as a reservoir of transforming growth factor beta (TGF-beta) in the extracellular milieu. We have examined the susceptibility of DCN to five different matrix metalloproteinases (MMPs): MMP-1 (tissue collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), MMP-7 (matrilysin) and MMP-9 (gelatinase B). MMP-2 and MMP-3 digest DCN into seven major fragments in a similar pattern. The N-terminal sequence of the two fragments generated by MMP-2 and MMP-3 is Leu211-Lys-Gly-Leu-Asn, but that of the others is Asp1-Glu-Ala-Ser-Gly. MMP-7 cleaves DCN into three major fragments which have the N-termini Asp1-Glu-Ala-Ser-Gly, Glu2-Ala-Ser-Gly-Ile and Leu244-His-Leu-Asp-Asn. Activities of MMP-1 and MMP-9 against DCN are negligible. The values of Km for the MMPs capable of degrading DCN are very similar (10-12 microM), but the kcat/Km value for MMP-7 (30.5 microM-1.h-1) is 4.5-fold higher than those for MMP-2 and MMP-3. Incubation of a DCN-TGF-beta1 complex with MMP-2, -3 or -7 results in release of TGF-beta1 from the complex. These data indicate proteolytic degradation of DCN by MMP-2, MMP-3 and MMP-7, and suggest the possibility that, under pathophysiological conditions, the digestion by the MMPs may induce tissue reactions mediated by TGF-beta1 released from DCN in the connective tissues.Biochemical Journal 04/1997; 322 ( Pt 3):809-14. · 4.65 Impact Factor
FEBS 15824 FEBS Letters 369 (1995) 249 251
Degradation of vitronectin by matrix metalloproteinases-1, -2, -3, -7 and -9
Kazushi ImaP, Hideo Shikata b, Yasunori Okada ~'*
~Department of Molecular Immunology and Pathology, Cancer Research Institute. Kana=awa University,
13-1 Takara-machi, Kanazawa, Ishikawa 920. Japan
bDepartment of Oral Pathology, School of Dentistry. Meikai UniversiO. Sakado. Saitama 350 ~)2. ,hlpan
Received 8 June 1995
Abstract The susceptibility of vitronectin (Vn) purified from
human plasma to digestion by matrix metalloproteinases
(MMPs) was examined. MMP-2, -3, -7 and -9 except for MMP-
1 degraded Vn into multiple fragments. MMP-7 showed the high-
est activity to the substrate among these MMPs, digesting 8-, 30-
and 44-fold more preferentially than MMP-2, -3 and -9, respec-
tively. These data suggest that MMP-2, -3, -7 and -9 may be
responsible for the pathological degradation and/or normal turn-
over of Vn.
2. Materials and methods
Materials were obtained as follows: Brij 35 and diisopropyl fluoro-
phosphate from Sigma Chemical Co.: acrylamide, ethylendiamine-
tetraacetic acid (EDTA) and SDS from Wako Chem., Japan; 4-amino-
phenylmercuric acetate (APMA) from Aldrich Chemical Co.: heparin-
Sepharose CL-6B from Pharmacia Biotech. The zymogens of MMP-I.
MMP-2, MMP-3, MMP-7 and MMP-9 were purified and activated by
incubation with APMA as described previously [6 9].
Key words. Vitronectin; Matrix metalloproteinase:
Vitronectin (Vn), an extracellular matrix component in vari-
ous tissues, was originally identified as serum protein secreted
from the liver . However, recent studies demonstrated the
expression of Vn mRNA in the extrahepatic tissues . Vn can
bind to plasminogen activator inhibitor type 1, plasminogen,
complement complex, perforin and antithrombin III in the cir-
culation, and to collagen and elastin microfibrils in extracellu-
lar milieu, suggesting that Vn may play important biological
roles in the tissues . Actually, Vn is a substrate for cellular
adhesion and stimulates motility of the keratinocytes and
smooth muscle cells, retinal neurite outgrowth, and migration
of the embryonal neural crest cells . In pathological condi-
tions, Vn may be implicated in the tumor cell invasion and
metastasis, since expression of the integrin type receptor for Vn
is well correlated with the malignant phenotype of tumor cells
such as melanoma, glioblastoma and lung carcinoma cells [4,5].
However, no information is so far available for the proteinases
involved in turnover and pathological degradation of Vn. In the
present study, we have examined susceptibility of Vn to five
different matrix metalloproteinases (MMPs) and found that it
can be digested by MMP-2 (gelatinase A; EC 184.108.40.206), MMP-
3 (stromelysin 1; EC 220.127.116.11), MMP-7 (matrilysin; EC
18.104.22.168) and MMP-9 (gelatinase B; EC 22.214.171.124), but not by
MMP-I (tissue collagenase; EC 126.96.36.199).
2.2. Purf/qcation ~!' vitronectin
Purification protocol of Vn fiom human plasma was essentially the
same as reported by Yatohgo et al. . Human plasma was applied
to heparin-Sepharose column chromatography equilibrated with 50
mM Tris HC1, pH 7.5, and 0.15 M NaC1. The flow-through fractions
were treated with 8 M urea and applied to the column equilibrated with
8 M urea in the buffer. Vn bound to the column was eluted with 50 mM
Tris-HC1, pH 7.5, 0.5 M NaC1, 5 mM EDTA and 8 M urea. The
purified Vn showed a doublet of 75,000- and 65,000-M, bands as re-
ported . It was then dialyzed against 50 mM Tris HC1, pH 7.5, 0.15
M NaCI, 0.05% Brij 35 and 0.02% NAN3. Alter addition of 2 mM
diisopropyl fluorophosphate. Vn was stored at -20°C until used.
2.3. Degradation O/Vn by MMPs
Digestion of Vn was first carried out by incubation of the substrate
at 37°C for up to 24 h with each MMP in an enzyme-to-substrate ratio
of 1:30 in 50 mM Tris HC1, pH 7.5, containing 0.15 M NaCI, 10 mM
CaC12, 0.05% Brij 35 and 0.02% NaN 3. To show the digestion frag-
ments, MMP-I, -2, -3, -7 and -9 were incubated t\~r 4 h at 37°C with
the substrate in enzyme-to-substrate ratios of 1: 10. 1:20, 1:10, 1:700
and 1: 10, respectively. The reactions were terminated with 20 mM
EDTA and the products were analyzed by SDS-polyacrylamide gel
electrophoresis (PAGE) (10% acrylamide) under reducing conditions.
To determine catalytic efficiency of MMP-2, -3, -7 or -9, Vn ( 10/~g)
was incubated for 2 h at 37°C with each APMA-activated MMP in a
final reaction mixture of 70/~1 (50 mM Tris-HC1, pH 7.5, 0.15 M NaCI,
10 mM CaCI2, 0.05% Brij 35 and 0.02% NAN3). Seven different concen-
trations of each MMR i.e. enzyme-to-substrate ratios of 1:25 I :150 for
MMP-2, -3 and -9 and 1:400-1:1000 for MMP-7, were used for the
study. After SDS-PAGE, the protein bands in the gels were stained with
1% Coomassie Brilliant Blue and densitometrically scanned using com-
puter assisted image analysis. The reaction velocity was quantitated by
measuring the disappearance of 75,000- and 65,000-M,. bands of intact
Vn, since the single cleavage product of Vn was not obtained. The
disappearance patterns were linear with time until approximately 20
25% of the substrate was degraded. Based on the molecular masses o1"
51,929, 70,952, 52,220, 27,938 and 78,426 for MMP-1, -2, -3, -7 and -9,
respectively, the extinction coefficients were calculated . F~' ~'; = 1.3,
1.9. 1.1, 1.6 and 1.3 ml/mg for MMP-1, -2, -3, -7 and -9. respectively,
were used l\~r the studies .
*Corresponding author. Fax: (81) (762) 34-4508.
E-mail: yasokada@icews 1 .ipc.kanazawa-u.ac.jp
Abbreviations." APMA, 4-aminophenylmercuric acetate; EDTA, ethyle-
nediaminetetraacetic acid; MMP, matrix metalloproteinase; PAGE,
polyacrylamide gel electrophoresis; Vn, vitronectin.
2.4. Determination q[' K,, and V ......
Km and l~;n, X of Vn degradation of MMPs were determined using the
methods described by Welgus et al. . Briefly, increasing amounts of
Vn (8, 10, 12, 14 and 16/lg) were incubated with 1.6/lg of MMP-9, 400
ng of MMP-2 and MMP-3, or 16 ng of MMP-7 for 2 h at 37°C. The
reaction was performed in 50 mM Tris-HC1, pH 188.8.131.52 M NaCI, 10
mM CaCI 2, 0.05% Brij 35 and 0.02% NaN, at a final volume of 75/A.
The reaction mixtures were subjected to SDS-PAGE under reduction
0014-5793/95/$9.50 © 1995 Federation of European Biochemical Societies. All rights reserved.
and the initial velocity of disappearance of the 75,000- or 65,000-M r
bands in the gels was measured densitometrically as described above.
Proteolytic fragments of human plasma Vn by MMP-1, -2,
-3, -7 and -9 were analyzed on SDS-PAGE under reduction.
Among these MMPs, MMP-2, -3, -7 and -9 but not MMP-1
digested Vn (Fig. 1). MMP-2, -3 and -9 digested it into similar
major fragments ranging from 62,500- to 31,500-M r, although
some minor bands were different from each other. These prod-
ucts were resistant to further degradation even after longer
incubation up to 24 h in a high enzyme-to-substrate ratio of
1:10 (data not shown). On the other hand, MMP-7
showed different digestion products ranging from 61,500- to
22,500-M,. (Fig. 1). Longer incubation of the reaction mixture
degraded these fragments into smaller peptides and only the
digestion product of 22,500-M~ species remained after 24 h incu-
bation (data not shown). These results suggest that MMP-7 has
the strong proteolytic activity to Vn and cleaves it at different
sites of the substrate from those by MMP-2, -3 and -9.
The efficiency of the Vn-degrading MMPs was examined by
incubation of Vn with different concentrations of the MMPs.
Table 1 shows the relative catalytic efficiency of MMP-2, -3, -7
and -9 to cause 50% cleavage of the substrate. Vn was com-
posed of 75,000- and 65,000-Mr species in a ratio of 3.5:6.5 by
densitometrical analysis. Both species were equally susceptible
to digestion by all the MMPs used in this study. MMP-7 was
the most effective enzyme; the efficiency was about 8-, 30- and
44-fold higher than MMP-2, -3 and -9, respectively.
The kinetic parameters, Km and K~.at, were determined for
MMP-2, -3, -7 and -9. Since a single initial proteolysis could not
be obtained by lowering enzyme concentrations or tempera-
ture, the stained gels were scanned densitometrically for the
disappearance of intact Vn molecule. In these experiments,
degradation of Vn was always kept to < 25% to measure initial
velocity, even at the lowest concentration of Vn employed and
graphed by the method of Lineweaver and Burk. K,. of each
1 2 3 4 5 6 7
Fig. 1. SDS-PAGE of the reaction products of Vn by incubation with
MMP-1, -2, -3, -7 and -9. The substrate (10 pg) was incubated with
MMP-1 (1 pg, lane 2), MMP-2 (0.5 fig, lane 3), MMP-3 (1 fig, lane 4),
MMP-7 (14 ng, lane 5), or MMP-9 (1 ~ug, lane 6) for 4 h at 37°C. After
termination of the reaction with 20 mM EDTA, the digestion products
were subjected to SDS-PAGE (I0% acrylamide) under reduction and
stained with 1% Coomassie Brilliant Blue. Lanes 1 and 7 are the control
samples incubated with buffer alone for 0 and 4 h, respectively.
K. lmai et aL/FEBS Letters 369 (1995) 249 251
Catalytic efficiency of vitronectin by matrix metalloproteinases
Mol of enzyme producing 50%
degradation of vitronectin
MMP-2 4.6 x 10 -l-'
MMP-3 1.5 x IW u
MMP-7 5.2 x 10 -I~
MMP-9 2.3 x 10 -I~
75,000-M r form
3.7 x 10 -12
1.5 x 10 <l
4.7 x l0 <~
2.1 x 10 -It
Each metalloproteinase was incubated with vitronectin (10,ug) at 37°C
for 2 h using different enzyme concentrations as described in section
2. Degradation was extrapolated linearly to estimate the number of
metalloproteinase required to produce 50% substrate cleavage.
MMP for both 65,000- and 75,000-M r species of Vn was ap-
proximately equal (Table 2). Values for K~,, were observed
within the range of 0.03 1.2 molecules of 65,000-M r Vn/mole-
cule of MMP/min and 0.02 1.1 molecules of 75,000-M,. Vn/
molecule of MMP/min. K~,,t/K,,, an index of specific activity,
ranged from 2.0 x 104 M I rain -t to 0.9 x 106 M ~ rain < for the
65,000-M,. species and for 2.0 x l0 4 M ~ rain < to 1.1 x 106 M
m in-t tbr the 75,000-M r species. The value of Kc~,/K,, of M M P-7
was approximately 11-, 30- and 50-fold higher than that of
MMP-2, -3 and -9, respectively.
The present study is the first to demonstrate the proteolytic
degradation of Vn. Among five MMPs used, i.e. MMP-I, -2,
-3, -7 and -9, all but MMP-1 cleaved Vn, suggesting that Vn is
a susceptible substrate of the MMP family members. Although
MMP-3 and -7 share the substrates, degradation products of
the extracellular matrix macromolecules such as fibronectin
and laminin by the enzymes are dissimilar [8,12] and the diges-
tion patterns of Vn are also different. Recent studies have
indicated that MMP-7 has the highest activity against cartilage
link protein, aggrecan, entactin, insoluble elastin, and tenascin
[8,13 17]. The present study further demonstrates that MMP-7
is also the most effective enzyme for Vn digestion with 8-, 30-
and 44-fold higher activities than MMP-2, -3 and -9, respec-
tively. The data showing that the values of Km of MMP-2, -3,
-7 and -9 against 65,000- or 75,000-M r species of Vn
(1.0 ~ 1.5 x 10 6 M) are almost identical indicate similar bind-
ing activities of these MMPs to Vn. It is definite that the high
specific activity of MMP-7 to Vn is ascribed to its catalytic
activity. However, the precise molecular mechanism is un-
known at the present time. It may be possible to speculate that
this is because MMP-7 which lacks the COOH-terminal Vn-like
domain can readily access the substrate. MMP-2 and -9 are
structurally related and share the substrates such as gelatins
and type IV and V collagens. However, our previous studies
showed that MMP-2 degrades glycoproteins including laminin
and fibronectin while MMP-9 cleaves type III collagen and ~2
chain of type I collagen [6,9]. For the degradation ofVn MMP-
2 was more effective enzyme than MMP-9, but both showed
similar digestion products, suggesting the cleavage at the simi-
lar sites. MMP-1 was considered to exclusively hydrolyze fibril-
lar collagens, but recent studies have shown that it also digests
other proteins including aggrecan, entactin, cartilage link pro-
IC lmai el ul /blEBS Letters 369:19951 249 251
Kinetic parameters of MMPs on Vn
Metalloproteinase K m (M)
(tool of vitronectin digested/tool
(M 7min ~)
65,000-M r lk~rm
of x itronectin
1.2 x 10 ~'
1.5 x 1(1 ~'
1.4x 10 ~'
1.5 × I0 "
l.O x 10 6
1.4x 10 <'
1.0x 10 ~'
1.3 x I0 -~'
8.0 x 1(14
3.4 x 104
2.0 ,< l(P
I.O × 10'
3.5 × lit'
1.1 × I(I ~'
231 × I()'
The K,, and K,~,, of metalloproteinases on both 65,000- and 75,000-M, species ofxitronectin were determined as described in section 2 The ratio K~.,,/K,,,
is used as an index of specitic activity.
tein, and tenascin [13 15,17], However, Vn is not a substrate
for the enzyme.
Vn is known to be localized in the loose connective tissue,
dermis, and atherosclerotic arterial wall . Because of the
wide distribution in eleastin-containing tissues and the proper-
ties to interact with other extracellular matrix proteins, it is
considered to serve as a molecular link between elastic fibers
and the surrounding collagen/proteoglycan scaffold . Vn is
also a ligand for cell adhesion through integrins, urokinase
receptor and/or cell surfitce-associated proteoglycan . The
expression of the intergrins for Vn in tumor cells is closely
related with their invasive and metastatic phenotype . In the
current study, we have shown that MMP-2, -3, -7 and -9 de-
grade Vn. Since these MMPs are expressed in the tissues of
artherosclerotic region, wound healing and cancer , they
would degrade Vn under such pathological conditions. Actu-
ally, it has been reported that Vn extracted from the human
adult dermis is composed of the degraded fragments with M~
58,000, 50,000, 42,000, 35,000 and 27,000 , which appear to
be similar to those obtained by digestion with MMPs. In addi-
tion, Seftor et al.  reported that treatment of melanoma cells
with Vn or antibodies to the Vn receptor induces MMP-2
expression, enhancing cellular invasion through reconstituted
basement membrane. Thus, it seems likely that MMPs may
participate in the degradation and/or turnover of Vn in the
tissues under the pathophysiological conditions and also mod-
ulate tumor cell inwtsion through degradation of Vn.
Ackmnrh'~&ements: We are grateful to Dr. Bj6rn Dahlb~ck of the
Department of Clinical Chemistry, the University of Land, Sweden, t\)r
providing us with vitronectin in the earlier experiments and helpful
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