Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Transferases
Reexamination Certificate
1999-01-19
2001-04-17
Nashed, Nashaat T. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Transferases
C435S002000, C435S232000, C435S252330, C435S320100, C536S023200
Reexamination Certificate
active
06217863
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to polysaccharide lyases and the rational design of the same. In particular, the present invention relates to new polysaccharide lyases rationally designed and based upon the heparinase I of
Flavobacterium heparinum.
The polysaccharides heparin and heparin sulfate are characterized by a disaccharide repeating unit of uronic acid and hexosamine, where the uronic acid is either L-iduronic acid or D-glucuronic acid and the glucosamine is linked to the uronic acid by a 1→4 linkage (Jackson et al., 1991). Heparin-like molecules are complex due to the high degree and varying patterns of sulfation on both the uronic acid and the hexosamine residues. It is believed that it is the sulfation which is responsible for the numerous different functional roles of these carbohydrates. Our understanding of heparin's functional role is severely limited by our poor knowledge of the heparin sequence.
Heparinases have proved to be useful tools in heparin degradation and in providing composition and sequence information (Linhardt et al., 1990).
F. heparinum
produces at least three types of heparinases (I, II and III) with different substrate specificities (Lohse & Linhardt, 1992). It has been proposed that all three enzymes cleave heparin through an elimination reaction catalyzed by a nucleophilic amino acid.
Heparinase I (or heparin lyase I, EC 42.2.7) is a 42,500 Da enzyme isolated from the periplasm of
F. heparinum
which cleaves heparin specifically in a random endolytic fashion (Linker and Hoving, 1972; Linhardt et al., 1982) at linkages of the type H
NS,6X
−
I
2S
or H
NS,6X
−
I
2X
, where X is either sulfated or unsubstituted (Linhardt, et al., 1990; Desai, et al., 1993). The characteristic heparin degradation product profile includes &Dgr;U
2S
H
NS
(disaccharide 1); &Dgr;U
2S
H
NS,6S
(disaccharide 2), &Dgr;U
2S
H
NS
I
2S
H
NS,6S
(tetrasaccharide 1), &Dgr;U
2S
H
NS,6S
GH
NS,6S
(tetrasaccharide 2), &Dgr;U
2S
H
NS,6S
I
2S
H
NS,6S
(tetrasaccharide 3), and &Dgr;U
2S
H
NS,6S
IH
NAc,6S
GH
NS,3S,6S
(hexasaccharide).
Heparinase I has recently been cloned and expressed in
E. coli
(Sasisekharan et al., 1993). The enzyme has be utilized in the sequence determination of sugars, in the preparation of small heparin fragments for the therapeutic uses, and in the ex vivo removal of heparin from blood (Linhardt et al., 1990; Bernstein et al., 1988). Extracorporeal medical devices (e.g. hemodialyzer, pump-oxygenator) rely on systemic heparinization to provide blood compatibility within the device and a blood filter containing immobilized heparinase I at the effluent which is capable of neutralizing the heparin before the blood is returned to the body (Bernstein et al., 1988).
It has been suggested that heparinase I binds heparin through lysine residues on the enzyme surface (Yang et a., 1985; Linhardt et al., 1982). The importance of lysines in enzyme activity is suggested by the observation that modification by amine-reactive reagents and immobilization of heparinase I on amine-active supports result in extensive activity losses (Comfort et al., 1989; Leckband & Langer, 1991; Bernstein et al., 1988). Further evidence for an electrostatic nature of the interaction lies in the pH and ionic strength dependence of heparinase activity (Yang et al., 1985). Additionally, the finding that tetrasaccharides are the smallest heparin fragments that still retain substrate activity gives some information about the size requirements of the active site (Linhardt et al., 1990). Despite these observations, information concerning the structure of the enzyme has been scant.
There has been much speculation in the art about the possibility of creating “designer” enzymes, rationally designed to have desired substrate specificities and activities, and heparinase I would be an appropriate staring point for the rational design of novel polysaccharide lyases. Yet, although the importance of different levels (primary, secondary, and tertiary) of protein structure in determining the functional activity of enzymes has long; been recognized, the lack of a broad and detailed understanding of the relationship be structure and function has prevailed significant progress. Even for enzymes which have known activities, substrates, and primary structure, the general lack of information about secondary and tertiary structures and the relationship of these to function has made it difficult to predict the functional effect of any particular changes to the primary structure.
SUMMARY OF THE INVENTION
The present invention provides for new polysaccharide lyases derived from heparinase and rationally-designed based upon detailed structural and functional characterization of heparinase. In particular, in one series of embodiments, the present invention provides substantially pure polysaccharide lyases composing the amino acid sequence of the mature heparinase I protein of
F. heparinum
in which at least one amino acid residue has been substituted and in which the substitution is (a) a substitution of a cysteine residue corresponding to position 135 of SEQ ID NO: 2 with a residue selected from the group consisting of aspartate, glutamate, serine, threonine, and histidine; (b) a conservative substitution of a residue of a Cardin-Weintraub-like heparin-binding sequence XBBXXXBXB (SEQ ID NO: 3) corresponding to positions 197-205 or 208-212 of SEQ ID NO: 2 with a residue which conforms to the heparin-binding sequence; (c) a conservative substitution of a residue of an EF-hand-like calcium binding sequence corresponding to positions 206-220 of SEQ ID NO: 2 with a residue which conforms to the calcium binding sequence; (d) a conservative substitution of a residue of a PB1, PB2 or PB3 &bgr;-sheet domain of SEQ ID NO: 2; (e) a non-conservative substitution of a cysteine residue corresponding to position 297 of SEQ ID NO: 2; (f) a non-conservative substitution of a residue of a PB1, PB2 or PB3 &bgr;-sheet domain of SEQ ID NO: 2 which preserves a parallel &bgr;-helix tertiary structure characteristic of SEQ ID NO: 2;(g) a deletion of one or more residues of a N-terminal region or a C-terminal region of SEQ ID NO: 2 which preserves a parallel &bgr;-helix tertiary structure characteristic of SEQ ID NO: 2; or (h) a non-conservative substitution of a serine residue corresponding to position 39 of SEQ ID NO: 2.
The present invention thus contemplates any of the foregoing substitutions alone, but also contemplates combinations of these substitutions which result in functionally active modified heparinases with altered stability, activity, and/or specificity as described in greater detail below.
It is, for example, a particular object of the invention to provide substantially pure polysaccharide lyases based upon heparinase I in which the cysteine residue corresponding to position 135 of heparinase I has been substituted with an aspartate, glutamate, serine, threonine, or histidine.
It is, for example, another particular object of the invention to provide substantially pure polysaccharide lyases based upon heparinase I in which the serine residue corresponding to position 39 of heparinase I has been substituted with an alanine residue.
It is, for example, another particular object of the invention to provide substantially pure polysaccharide lyases based upon heparinase I in which a residue of a Cardin-Weintraub-like heparin binding sequence XBBXXXBXB corresponding to positions 197-205 or 208-212 of heparinase I has been conservatively substituted with a residue which conforms to the heparin binding sequence. In a preferred set of embodiments, the conservative substitution is of a lysine residue corresponding to position 198, 199 or 205 of heparinase I with an arginine or histidine, most preferably an arginine. In other preferred embodiments, the conservative substitution is of the histidine residue corresponding to position 203 of heparinase I.
It is, for example, yet another particular object of the invention to provide substantially pure polysaccharide lyases based upon heparinas
Cooney Charles L.
Ernst Steffen
Godavarti Ranganathan
Langer Robert
Sasisekharan Ram
Massachusetts Institute of Technology
Nashed Nashaat T.
Wolf Greenfield & Sacks P.C.
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