Gene encoding hyaluronan synthase

Details Gene encoding hyaluronan synthase Gene encoding hyaluronan synthase

1996-07-03

2002-12-10

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

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

C435S252300, C435S320100, C435S325000, C435S084000, C536S023200, C536S023100

Reexamination Certificate

active

06492150

ABSTRACT:

BACKGROUND OF THE INVENTION
Hyaluronan (HA, hyaluronic acid) is a linear unbranched polymer made up of repeating disaccharide units of D-glucuronic acid (&bgr;1→3) N-acetylglucosamine (&bgr;1→4). HA biosynthesis requires two enzyme activities; the transfer of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcUA), respectively, to the growing HA chain. HA is synthesized at the inner face of the plasma membrane and is subsequently extruded to the outside of the cell. HA is a major constituent of the extracellular matrix during embryonic development. For example, within the developing embryo, HA accumulates at sites of cell migration and proliferation, and has been proposed to play important roles in craniofacial, limb, neural tube, and heart development. In particular, HA is essential for the formation of endocardial cushions, the structures required for septation and the development of heart valves. In adults, HA is a major constituent of the extracellular matrix of most tissues and organs, and a critical component of the vitreous humor of the eye, joint fluid and cartilage.
HA is highly biocompatible and completely biodegradable, and has demonstrated beneficial effects when administered to the joints of arthritic race horses and to perforated rat tympanic membranes. HA has also been employed to protect eye tissue during artificial intraocular lens implantations, as a delivery agent for drugs and to prevent post-operative scarring.
Genes which encode HA biosynthetic enzymes have been identified in bacteria, e.g., Group A Streptococcus (Wessels et al.,
Infect. Immuh.
62, 433 (1994); DeAngelis et al.,
J. Biol. Chem.,
268, 19181 (1993); DeAngelis et al.,
Biochemistry,
33, 9033 (1994)). Polymerization of HA by
S. pyogenes
occurs through the action of a single enzyme, HA synthase, encoded by the hasA gene. The
S. pyogenes
HA synthase is localized to the membrane and is predicted to have several transmembrane domains and a large intracellular loop encompassing the active site of the enzyme. Purified immobilized HasA has been shown to be sufficient for HA polymerization in vitro (DeAngelis et al.,
Biochemistry,
33, 9033 (1994)). The transfer of the hasA gene and a second gene, hasB, into heterologous bacterial species results in the synthesis of an HA capsule (DeAngelis et al.,
J. Biol. Chem.,
268, 19181 (1993)). The hasb gene encodes a UDP-glucose dehydrogenase, which converts UDP-glucose to UDP-glucuronic acid (UDP-GlcUA), a subunit of HA.
However, there is evidence that other genes are also involved in bacterial HA biosynthesis. A protein originally identified in
Streptococcus equisimilis
as HA synthase (Lansing et al.,
Biochem. J.,
289, 179 (1993)) has no sequence similarity to
S. pyogenes
HasA but has significant sequence similarity to bacterial proteins involved in oligopeptide binding and transport. Although the total amount of HA synthesized by bacterial cells overexpressing the
S. equisimilis
HA synthase increased, the length of the resultant HA chains was significantly shorter, suggesting that the increase may be a function of an elevation in the rate of HA transport from the cell (O'Regan et al.,
Int. J. Biol. Macromol.,
16, 283 (1994)). Thus, rather than being directly involved in HA biosynthesis, the
S. equisimilis
HA synthase may be involved in the transport of HA, or may participate in HA synthesis as an accessory molecule, rather than as the synthase itself.
While both bacterial and animal sources of HA exist, high molecular weight HA is difficult and costly to isolate and purify due to the fact that HA is complexed with proteoglycans. Moreover, both bacterial and animal sources of HA are increasingly under more stringent regulatory controls due to fear of contamination with identifiable, or as yet unidentified, infectious or toxic agents. Furthermore, the extensive purification process of HA polymer from cells results in an HA polymer of considerable molecular weight polydispersity.
Thus, there is a need to isolate and purify genes that encode eukaryotic HA biosynthetic enzymes or proteins associated with the extracellular accumulation of HA.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified DNA molecule comprising a preselected DNA segment encoding eukaryotic, preferably mammalian, hyaluronan synthase-2 (Has2), or which encodes a biologically active subunit thereof. A preferred embodiment of the invention is a DNA molecule comprising a preselected DNA segment, e.g., SEQ ID NO:1, that encodes murine hyaluronan synthase-2. A murine hyaluronan synthase-2 having SEQ ID NO:2 has 21% identity and 28% similarity to Streptococcal HasA, and 55% identity and 73% similarity to murine Hasl (Itano et al.,
J. Biol. Chem.,
271, 9875 (1996)). Because the deduced amino acid sequence of Has1 is distinct from the murine hyaluronan synthase-2 having SEQ ID NO:2, there appears to be more than one mammalian gene encoding an enzyme or protein which is associated with HA biosynthesis and/or extracellular HA accumulation. Another preferred embodiment of the invention is a DNA molecule comprising a preselected DNA segment, e.g., SEQ ID NO:23, that encodes human hyaluronan synthase-2. Also provided is an isolated and purified DNA molecule comprising a preselected DNA segment which encodes a protein that increases the amount of extracellular hyaluronan produced by cultured primate cells transformed so as to express said DNA segment.
Further provided is an isolated and purified DNA molecule comprising a DNA segment encoding eukaryotic hyaluronan synthase-3. A preferred embodiment of the invention includes a DNA segment comprising SEQ ID NO:25 which encodes a human hyaluronan synthase-3 comprising SEQ ID NO:27. Another preferred embodiment of the invention includes a DNA segment comprising SEQ ID NO:26 which encodes a murine hyaluronan synthase-3 comprising SEQ ID NO:28. The DNA molecules of the invention are double-stranded or single-stranded, preferably, they are cDNA.
An isolated and purified DNA molecule, such as a probe or a primer, of at least seven, preferably at least fifteen, nucleotide bases which hybridizes under stringent conditions to the DNA molecules of the invention, or RNA molecules derived from these DNA molecules, is also provided by the invention. The term “stringent conditions” is defined hereinbelow. The probes or primers of the invention have at least about 80%, preferably at least about 90%, identity to the above-disclosed DNA sequences. A preferred embodiment of the invention includes a probe or primer which has at least about 80%, preferably at least about 90%, identity to 1) SEQ ID NO:25 or 2) SEQ ID NO:26. The probes or primers of the invention are detectably labeled or have a binding site for a detectable label. Such probes or primers are useful to detect, quantify and/or amplify DNA strands with complementary to sequences related to hyaluronan synthase-2 or hyaluronan synthase-3 in eukaryotic tissue samples.
The present invention also provides an expression cassette comprising a promoter which is functional in a host cell operably linked to a preselected DNA segment encoding hyaluronan synthase-2. Preferably, the expression cassette comprises a preselected DNA segment encoding murine hyaluronan synthase-2. Another preferred embodiment of the invention is an expression cassette comprising a preselected DNA segment encoding human hyaluronan synthase-2. Such expression cassettes can be placed into expression vectors which can then be employed to transform prokaryotic or eukaryotic host cells. The present vectors can also contain a functional DNA sequence which is a selectable marker gene or reporter gene, as described below.
Also provided is a transformed host cell, the genome of which has been augmented by a preselected DNA sequence encoding hyaluronan synthase-2. Preferably, the preselected DNA sequence is integrated into the chromosome of the transformed host cell, and is heritable.
Expression of mouse hyaluronan synthase-2 in COS-1 cultured primate cells results in the form

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