A NOVEL MATRIX METALLOPROTEASE-9 WHICH DEGRADES COLLAGEN AND EXISTS IN A PROTEOGLYCAN
FORM
*Patchigolla, R Krishna R.; **Burkhart, W A; +***Schmid, T M. (A-NIH)
*Rush University, Chicago, IL. +***Rush Medical Collage and Rush University, Chicago, IL. Department of Biochemistry/1653 W Congress Pkwy/Chicago, IL
60612, 312-942-3051, Fax: 312-942-3053, [email protected]
Introduction: Matrix metalloproteinases (MMPs) are neutral extracellular
enzymes that play a prominent role in tissue remodeling like endochondral
bone formation and degenerative conditions like arthritis. In ovo the
embryonic tibia develops initially as a cartilage model which is resorbed and
replaced by bone matrix. Chick embryonic tibias (CETs) were maintained in
organ culture as a model of cartilage degradation. The appearance of a high
molecular weight enzyme correlated with cartilage loss in these cultures [1,
2]. The enzyme was purified, partially sequenced and characterized. It is a
MMP with gelatinolytic activity and the ability to cleave triple helical
collagen. It exists primarily in a proteoglycan (PG) form with chondroitin
sulfate chains. Protein sequence data suggests it is the avian homolog of
MMP-9
Surprisingly, the high molecular weight of this enzyme is due to the presence
of covalently associated chondroitin sulfate chains. This 230 kDa enzyme
remained intact even after the enzyme was subjected to an equilibrium
ultracentrifugation in a CsCl2 gradient in the presence of 4 M GnHCl.
Moreover an 35S-sulfate label co-purified with the enzyme and was removed
by a protease-free chondroitinase digestion. The data suggest that the 230 kDa
enzyme is a PG. This is the first example of a MMP in a PG form. The GAG
chain on this MMP may affect its activity through binding to substrates or by
blocking inhibitor binding. Knock out experiments in mice show MMP-9 is
critical in the resorption of hypertrophic cartilage in the growth plate [3]. Our
experiments show the avian enzyme is capable of removing both triple-helical
collagen and denatured collagen molecules from this region.
Methods: Intact tibias from day 12 chick embryos were dissected and
cultured in Dulbecco’s modified Eagle’s medium (250 µl/tibia) with ascorbate
(50 µg/ml) and lippopolysacharide (LPS) (10 µg/ml). Conditioned media
(CM) were collected every 2 days and analyzed by zymograms. Pooled CM
were purified on a gelatin agarose affinity column and eluted with 0 to 10%
dimethylsulfoxide. Pooled affinity fractions containing the 230 kDa enzyme
were further purified on a DEAE sepharose column followed by elution with
0.15 to 1.0 M NaCl. This enzyme was shown to be a MMP by activation with
p-aminophenylmercuric acetate and by inhibition by
ethylenediaminetetraacetic acid or 1, 10-phenanthroline. The enzyme activity
was measured using radiolabeled 3H collagen or gelatin substrates and also
with fluorescent peptide substrates. To assess the glycosaminoglycan (GAG)
chains on the enzyme CET cultures were radiolabeled with 35S-sulfate,
purified by chromatography and analyzed by zymography and fluorography.
The intact enzyme was digested with chondroitinase to estimate the size of
protein or with papain to estimate the size of the GAG chains. The purified
protein was digested with endopeptidase Lys-C or trypsin and the peptides
products were sequenced by Edman degradation.
Sequenced Peptides
High sequence homolgy
DHFYWR
Bovine MMP-9
VLLFSGEHYWR
Bovine MMP-9
YDLLQCPQH
Human MMP-9
DSDDVFTGVPLDAR
Salamander MMP-9
VGTQGAFLIADTWPGLPAVI
Rabbit MMP-9
KVDPEVN
Mouse MMP-9
FDAITEITGELHFFK
Mouse MMP-9
Results: A high molecular weight (~230 kDa smear) gelatinase appeared in
the medium during the second weak of CET cultures, where as a 72 kDa
gelatinase was present through the culture period. Affinity purification of CM
resulted in a partial separation of 72 kDa and the 230 kDa gelatinolytic
activities. Subsequent DEAE chromatography completely separated the two
enzymes. The 72 kDa gelatinase did not bind to the DEAE column where as
the 230 kDa bound to it and was eluted with (~0.6M) NaCl. Incubation with
3
H collagen showed the 230 kDa enzyme had collagenase activity, but the 72
kDa gelatinase did not. Digestion of the 230 kDa enzyme with chondroitinase
resulted in a gel shift on zymograms from 230 kDa to about 80 kDa
suggesting the 230 kDa enzyme contained GAG chains. The migration of the
72 kDa enzyme did not change with similar treatments. Labeling of CM
proteins with 35S-sulfate showed the radiolabel was incorporated in the 230
kDa enzyme. This 35S label co-purified with the 230 kDa enzyme on gelatin
affinity chromatography and DEAE chromatography (Fig. 1). Sufficient
enzyme was purified from large scale cultures for protein sequencing. A
number of peptides from the Lys-C or tryptic digestion of the enzyme were
sequenced. Seven peptides showed a high degree of homology with MMP-9
from several different species (see table).
Discussion: Appearance of a 230 kDa enzyme correlates with the dramatic
loss of cartilage components in the LPS treated CET cultures. This enzyme
was purified by gelatin affinity and DEAE chromatography and was shown to
have gelatinase and collagenase activity. The protein sequence data from
several peptides of this enzyme suggest that this enzyme is the avian homolog
of MMP-9 (gelatinase-B). This is the first report of an avian MMP-9. Only
avian MMP-2, MMP-13 and MT1-MMP have been described previously.
1 2 eluted fractions.
1 2 eluted frations.
A) zymogram-affinity
B) Fluorogram-affinity
230
kDa
72
kDa
1
2 eluted fractions. 1 2 eluted frctions.
C) zymogram-DEAE
D) Fluorogram-DEAE
Fig.1: Purification gels of 230 kDa enzyme: A and B are gelatin affinity and
C and D are DEAE purification gels. All gelatinolytic enzymes can be seen as
clear bands on zymograms (A and C) while the 35S-sulfate label containing
molecules are the dark bands on fluorograms (B and D). For each gel, lane 1
is starting materiel (e.g. pooled CM for A and B or pooled affinity fractions
for C and D), lane 2 is unbound material and lanes 3-5 show eluted fractions.
This purification scheme separated the 230 kDa enzyme from the 72
gelatinase (lower doublet on zymograms) and demonstrated that 35S-sulfate
co-purified with 230 kDa enzyme.
References: 1. Cole, A. A. al. et 1993. Dev. Biol. 159, 528-534. 2. MikuniTakagaki, Y., and Cheng, Y. 1987. Arch. of Biochem. Biopys. 259 (2), 579588. 3. Vu, T. H. al. et 1998. Cell9 3, 411-422.
ACKNOWLEDGMENT: This work was supported by NIH (2P50-AR39239
and AG15650) and through a research collaboration agreement with the Glaxo
Wellcome Corporation.
**Glaxo Wellcome, Durham, NC.
Poster Session - New Investigator Recognition Awards - VALENCIA FOYER
0304
46th Annual Meeting, Orthopaedic Research Society, March 12-15, 2000, Orlando, Florida