Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reissue Patent
1999-03-29
2001-08-21
Zitomer, Stephanie W. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S252300, C435S252330, C435S253400, C435S320100, C435S849000, C435S885000, C435S193000, C536S023700
Reissue Patent
active
RE037336
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to
DNA encoding the enzyme hyaluronate synthase, and to the use of this DNA in the preparation of recombinant cells for the production of the hyaluronate synthase enzyme and/or hyaluronic acid
a method for providing hyaluronic acid.
2. Description of the Related Art
The incidence of Streptococcal infections is a major health and economic problem worldwide, particularly in the developing countries (21). One reason for this is due to the ability of Streptococcal bacteria to often grow undetected by the body's phagocytic cells (i.e., macrophages and polymorphonuclear cells (PMNs). These cells are responsible for recognizing and engulfing foreign microorganisms. One effective way by which the bacteria evade surveillance is by coating themselves with polysaccharide capsules, such as hyaluronic acid (HA) capsules. Since polysaccharides are generally nonimmunogenic, the encapsulated bacteria do not elicit an immune response and are, therefore, not targeted for destruction. Moreover, the capsule exerts an antiphagocytic effect on PMNs in vitro (
7
) and prevents attachment of Streptococcus to macrophages (
32
). Precisely because of this, in group A and group C Streptococci, the HA capsules are major virulence factors in natural and experimental infections (
9
,
10
). The group C Streptococcus equisimilis is responsible for osteomyelitis (
1
), pharyngitis (
2
), brain abscesses (
5
), and pneumonia (
20
,
25
).
Structurally, HA is a high molecular weight linear polysaccharide of repeating disaccharide units consisting of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA). HA is the only glycosaminoglycan synthesized by both mammalian and bacterial cells particularly groups A and C Streptococci. Some Streptococcus strains make HA which is secreted into the medium as well as HA capsules. The mechanism by which these bacteria synthesize HA is of interest since the production of the HA capsule is a very efficient and clever way by which Streptococci evade surveillance by the immune system.
HA is synthesized by both mammalian and Streptococcus cells by the enzyme hyaluronate synthase, which has been localized to the cytoplasmic membrane of Streptococcus (
14
). The synthesis of HA in these organisms is a multi-step process. Initiation involves binding of the first precursor, UDP-GlcNAc or UDP-GlcUA. This is followed by elongation which involves alternate addition of the two sugars to the growing oligosaccharide chain. The growing polymer is extruded across the bacterial plasma membrane and region of the cell wall and into the extracellular space. Although the HA biosynthetic system was one of the first membrane heteropolysaccharide synthetic pathways studied, the mechanism of HA synthesis is still not understood (
29
). This may be because in vitro systems developed to date are inadequate in that de novo biosynthesis of HA has not been accomplished. Chain elongation but not initiation occurs.
The direction of HA polymer growth is a matter of disagreement. Addition of the monosaccharides could be to the reducing (
17
) or nonreducing (
26
) end of the growing HA chain. In addition, other questions that need to be addressed are (i) whether nascent chains are linked covalently to a protein, to UDP or to a lipid intermediate, (ii) whether chains are initiated using a primer, and (iii) the mechanism by which the mature polymer is extruded through the plasma membrane of the Streptococcus. Understanding the mechanism of HA biosynthesis may allow development of alternative strategies to control Streptococcal infections by interfering in the process.
S. equisimilis strain D181 synthesizes and secretes HA. Investigators have used this strain and a group A strain A
111
to study the biosynthesis of HA and to characterize the HA-synthesizing activity in terms of its divalent cation requirement (
26
), precursor (UDP-GlcNAc and UDP-GlcUA) utilization (
8
,
13
), optimum pH (
26
), and molecular weight (
18
). Although a 52-kD protein has been identified as the HA synthase (
18
), no one has successfully purified to homogeneity an active enzyme. Moreover, it's not clear whether this molecule is all that is needed for the generation of hyaluronic acid, or whether it might act in concert with other cellular components or subunits. Thus, totally ex vivo methods of producing HA have not been forthcoming.
Typically, HA has been prepared commercially by isolation from either rooster combs or extracellular media from Streptococcal cultures. One method which has been developed for preparing HA is through the use of cultures of HA-producing streptococcal bacteria U.S. Pat. No. 4,517,295, describes such a procedure, wherein HA-producing Streptococci are fermented under anaerobic conditions in a CO
2
-enriched growth medium. Under these conditions, HA is produced and can be extracted from the broth. It is generally felt that isolation of HA from rooster comb is laborious and difficult, since one starts with HA in a less pure state. The advantage of isolation from rooster comb is that the HA produced is of higher molecular weight. However, preparation of HA by bacterial fermentation is easier, since the HA is of higher purity to start with. Usually, however, the molecular weight of HA produced in this way is smaller than that from rooster combs. Therefore, a technique that would allow the production of high molecular weight HA by bacterial fermentation Would be an improvement over existing procedures.
High molecular weight HA has a wide variety of useful applications—ranging from cosmetics to eye surgery. Due to its potential for high viscosity and its high biocompatability, HA finds particular application in eye surgery as a replacement for vitreous fluid. HA has also been used to treat racehorses for traumatic arthritis by intra-articular injections of HA, in shaving cream as a lubricant, and in a variety of cosmetic products due to its physiochemical properties of high visocity and its ability to retain moisture for long periods of time. In general, the higher molecular weight the HA that is employed the better. This is because HA solution viscosity increases with the average molecular weight of the individual HA polymer molecules in the solution. Unfortunately, very high molecular weight HA, such as that ranging up to 10
7
, has been difficult to obtain by currently available isolation procedures.
To address these or other difficulties, there is a need for new methods and constructs which can be used to produce HA having one or more improved properties such as greater purity or ease of preparation. In particular, there is a need to develop methodology for the production of larger amounts of relatively higher molecular weight and purity HA than is available from current technology. The present invention addresses one or more shortcomings in the art through the application of recombinant DNA technology.
SUMMARY OF THE INVENTION
The present invention involves the application of recombinant DNA technology to solving one or more problems in the art of hyaluronic acid preparation. These problems are addressed through the isolation and use of a
recombinant
DNA segment
encoding all or a portion of the hyaluronate synthase gene, the gene responsible for HA chain biosynthesis
identified in FIG.
5
. The
gene
recombinant segment
is cloned from DNA of an appropriate tissue or cellular source and engineered into useful recombinant constructs
for the preparation of HA and for the preparation of large quantities of the HA synthase enzyme itself
.
Through the application of techniques set forth herein, those of skill in the art will be enabled to obtain
a recombinant
DNA
segments encoding all or a portion of the HA synthase gene
segment identified in FIG.
5
. Through isolation of
the HA gene
this DNA,
from whatever source is chosen, one will have the ability to realize significant advantages such as an ability to manipulate the host that is chosen to express the
HA synthase gene
recombinant DNA segmen
Papaconstantinou John
Weigel Paul H.
Dunlap Codding & Rogers P.C.
The Board of Regents of the University of Oklahoma
Zitomer Stephanie W.
LandOfFree
Method for providing hyaluronic acid does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for providing hyaluronic acid, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for providing hyaluronic acid will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2487658