Molecular Cloning and Characterization of a Putative Mouse Hyaluronan Synthase

doi:10.1074/jbc.271.38.23400

Molecular Cloning and Characterization of a Putative Mouse Hyaluronan Synthase*

From the Department of Biochemistry and Molecular Biology, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259

^‡ To whom correspondence should be addressed:
Dept. of Biochemistry and Molecular Biology, Samuel C. Johnson Medical Research Bldg., Mayo Clinic Scottsdale, Scottsdale, AZ 85259
. Tel.: 602-301-8859; Fax: 602-301-7017.

Abstract

We report the isolation of a novel mouse gene which encodes a putative hyaluronan synthase. The cDNA was identified using degenerate reverse transcriptase-polymerase chain reaction. Degenerate primers were designed based upon an alignment of the amino acid sequences of Streptococcus pyogenes HasA, Xenopus laevis DG42, and Rhizobium meliloti NodC. A mouse embryo cDNA library was screened with the resultant polymerase chain reaction product, and multiple cDNA clones spanning 3 kilobase pairs (kb) were isolated. The open reading frame predicted a 63-kDa protein with several transmembrane sequences, multiple consensus phosphorylation sites, and four putative hyaluronan binding motifs. The amino acid sequence displayed 55% identity to mouse HAS, 56% identity to Xenopus DG42, and 21% identity to Streptococcus HasA. Northern analysis identified transcripts of 4.8 kb and 3.2 kb, which were expressed highly in the developing mouse embryo and at lower levels in adult mouse heart, brain, spleen, lung, and skeletal muscle. Transfection experiments demonstrated that mouse Has2 could direct hyaluronan coat biosynthesis in transfected COS cells, as evidenced by a classical particle exclusion assay. These results suggest that mammalian HA synthase activity is regulated by at least two related genes. Accordingly, we propose the name Has2 for this gene.

Footnotes

↵* This work was supported by Grant-in-Aid Postdoctoral Fellowship AZFW-7-95 from the American Heart Association, Arizona Affiliate (to A. P. S.), and by funds from the Mayo Foundation for Education and Research. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U52524[GenBank].
↵¹ The abbreviations used are:

HA

hyaluronan

RT-PCR

reverse transcriptase-polymerase chain reaction

ORF

open reading frame

CMV

cytomegalovirus promoter

PBS

phosphate-buffered saline

UTR

untranslated region

PKC

protein kinase C

PKA

protein kinase A

UDP-GlcNAc

UDP-N-acetylglucosamine

UDP-GlcUA

UDP-glucuronic acid

dpc

days postcoitum

bp

base pair(s)

kb

kilobase(s)

GAPDH

glyceraldehyde-3-phosphate dehydrogenase.
↵² A. P. Spicer and J. A. McDonald, manuscript in preparation.
- Received April 3, 1996.
- Revision received May 23, 1996.