Catalytically-active gelatinase mutant

Details Catalytically-active gelatinase mutant Catalytically-active gelatinase mutant

1995-05-19

2001-02-06

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S183000

Reexamination Certificate

active

06184021

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates to a truncated mutant of the 92 kDa gelatinase which is catalytically active comparable to the full protein but, unlike the full protein, is essentially inactive against insoluble elastin.
The truncated mutant of the invention is useful for treatment of disorders requiring the removal of excess connective tissue, e.g., keloids, post-operative fibrosis, intervertebral disc injections, hypertrophic scars, wound debridement, post-surgical adhesions and various fibrotic diseases (scleroderma, idiopathic pulmonary fibrosis) and the like treatments.
(Note: Literature references on the following background information and on conventional test methods and laboratory procedures well known to the ordinary person skilled in the art, and other such state-of-the-art techniques as used herein are indicated in parentheses, and appended at the end of the specification.)
Elastin is an extracellular matrix protein composed of hydrophobic tropoelastin monomers that are highly crosslinked. Elastin provides resilience to elastic fibers. The hydrophobicity of tropoelastin monomers, as well as their extensive crosslinking, result in an insoluble elastic fiber which is highly resistant to proteolysis (1). In the normal physiologic state, elastin undergoes minimal turnover (2). However, pathologic situations exist including pulmonary emphysema (3) and abdominal aortic aneurysm (4) which are characterized by proteolytic destruction of elastic fibers.
The matrix metalloproteinases comprise a gene family of enzymes that collectively are capable of degrading all components of extracellular matrix in physiologic and pathologic states (5). These enzymes are organized into homologous domain structures, with some differences in number of domains. All members share a zymogen domain and a zinc-binding catalytic domain. As presently recognized, this family consists of fibroblast, neutrophil, and a breast carcinoma-derived (6) collagenase, a 92 kDa gelatinase (also called gelatinase B and MMP-9), a 72 kDa gelatinase (also called gelatinase A and MMP-2), three stromelysins, macrophage metalloelastase, matrilysin (also known as PUMP and MMP-7), and a recently described 66 kDa membrane-type metallproteinase (7). Four members of this gene family have the capacity to degrade insoluble elastin. These are the 92 kDa gelatinase (8,9), the 72 kDa gelatinase (8,9), matrilysin (9), and macrophage metalloelastase (10,11).
The issue of substrate specificity has received considerable attention recently in matrix metalloproteinase biology. The structural determinants within these enzymes which confer the ability to degrade various substrates appear to be localized within discrete domains. For example, the ability of the collagenases to degrade triple-helical collagen requires the presence of the C-terminal hemopexin-like domain (12-14). In contrast, the stromelysins degrade a variety of substrates in a manner which is independent of the C-terminal hemopexin-like domain (13, 15-17). Unique to the 72 kDa and 92 kDa gelatinases is an additional domain composed of three fibronectin type II repeats inserted in tandem within the zinc-binding catalytic domain. This fibronectin-like domain is required for the gelatinases to bind efficiently to type I gelatin and type IV collagen (18-21). Matrilysin is the simplest member of this family of enzymes in that it contains only a zymogen domain and a catalytic zinc-binding domain.
A 92 kDa type IV collagenase (gelatinase) and the cDNA clone representing the full-length protein is disclosed in U.S. Pat. No. 4,992,537.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the present invention a truncated mutant of the 92 kDa gelatinase is provided which is catalytically active but essentially inactive against elastin. The truncated mutant preferably comprises residues 107-216 fused to residues 391-443 of the parent molecule.
This mutant representing the catalytic domain of the 92 kDa gelatinase has enzymatic activity against the conventional thiopeptolide substrate Ac-Pro-Leu-Gly-S-Leu-Leu-OEt comparable to the full-length 92 kDa gelatinase, and binds tissue inhibitor of metalloproteinases (TIMP) on an equimolar basis. It degrades gelatin but, surprisingly, it is inactive against insoluble elastin. The 92 kDa gelatinase, therefore, is unexpectedly unlike several other members of the matrix metalloproteinase family of enzymes such as matrilysin or macrophage metalloelastase in which the catalytic domains do account for elastolytic activity of these enzymes.
In accordance with a preferred embodiment of the invention, the catalytic zinc-binding domain of the 92 kDa gelatinase (92 CD) was expressed in
E. coli.
This gelatinase mutant was constructed such that it lacked the three fibronectin type II repeats contained within the catalytic domain of the native form of the enzyme. These internal fibronectin repeats were deleted by conventional polymerase chain reaction (PCR) procedures and the 92 CD was subcloned into a suitable expression vector for expression in
E. coli.
The resulting fragment coded for a protein lacking residues 217-390 of the parent molecule which encompass the three fibronectin-like repeats. A stop codon was placed after residue 443 to eliminate the collagen type V and hemopexin-like domains. The truncated mutant contained an additional Met-Gly dipeptide at the N-terminus in order to start translation at this point within the cDNA.
The protein sequence of the 92 CD, using the three-letter abbreviations of the constituent amino acid residues, is as follows:
Phe Gln Thr Phe Glu Gly Asp Leu Lys Trp His His His Asn Ile
[SEQ ID NO:1]
               5                   10                  15

Thr Tyr Trp Ile Gln Asn Tyr Ser Glu Asp Leu Pro Arg Ala Val
                20                  25                  30

Ile Asp Asp Ala Phe Ala Arg Ala Phe Ala Leu Trp Ser Ala Val
                35                  40                  45

Thr Pro Leu Thr Phe Thr Arg Val Tyr Ser Arg Asp Ala Asp Ile
                50                  55                  60

Val Ile Gln Phe Gly Val Ala Glu His Gly Asp Gly Tyr Pro Phe
                65                  70                  75

Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe Pro Pro Gly Pro
                80                  85                  90

Gly Ile Gln Gly Asp Ala His Phe Asp Asp Asp Glu Leu Trp Ser
                95       &e

Affiliated with

Also associated with

Rating

Catalytically-active gelatinase mutant does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Catalytically-active gelatinase mutant, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Catalytically-active gelatinase mutant will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2568303