(Received for publication, January 16, 1996; and in revised form, February 12, 1996)
From the
We developed a mammalian transient expression system to isolate
cDNA clones that determine hyaluronan expression.
HAS, a mouse mammary carcinoma mutant cell line,
which is defective in hyaluronan synthase activity, was first
established and used as a recipient for the expression cloning. One
cloned cDNA that overcame the deficiency was isolated. The cDNA termed
HAS contains an open reading frame of 1749 base pairs encoding a new
protein of 583 amino acids. Homology analysis of the amino acid
sequence suggests that HAS protein is related to streptococcal
hyaluronan synthase and also to Xenopus laevis DG42 protein
that was found to be homologous to bacterial hyaluronan synthase.
Expression of HAS cDNA in HAS
cells complemented not
only their mutant phenotypes such as deficient hyaluronan-matrix
deposition but also hyaluronan synthase activity itself. Therefore, HAS
cDNA is responsible for the activity of the hyaluronan synthase, a key
enzyme of hyaluronan synthesis in eukaryotic cells.
Hyaluronan, a high molecular weight linear glycosaminoglycan,
which is composed of 1,4-linked repeating disaccharides of
glucuronic acid
1,3-linked to N-acetylglucosamine, is a
characteristic component of the extracellular matrix during early
stages of morphogenesis, and its synthesis is spatially and temporally
regulated(1) . The association of hyaluronan with the cell
surface can influence the cellular behaviors especially in regard to
modulation of cell migration, adhesion, wound healing, and tumor
invasion(2, 3, 4, 5) . We are
interested in the molecules involved in the association and have found
that the heavy chain of the inter-
-trypsin inhibitor and
PG-M/versican play important roles in the formation of the hyaluronan
matrix(6, 7, 8) . The molecular cloning of
genes encoding enzymes that take part in the hyaluronan biosynthesis is
one of essential steps to understand the biosynthetic pathway as well
as to investigate biological functions of the pericellular association
of hyaluronan. Although the hyaluronan biosynthesis in Group A Streptococci has been extensively studied and the structural
gene for the bacterial hyaluronan synthase was recently isolated from Streptococcus pyogenes(9) , little is known about the
mechanism for the biosynthesis of hyaluronan in eukaryotic cells. There
are some attempts to purify eukaryotic hyaluronan synthase to a
homogeneity. However, those encountered the loss of enzyme
activity(10, 11, 12) . Therefore, we chose
the different way by adopting a mammalian transient expression system
to isolate the genes responsible for the expression of hyaluronan. We
first established several mutants that are defective in hyaluronan
biosynthesis. One of the mutant cell line, HAS
, is
defective in hyaluronan synthase activity and may therefore be useful
to identify a gene encoding eukaryotic hyaluronan synthase. In this
report, we describe the isolation of a cDNA encoding a protein that may
correspond to hyaluronan synthase in mouse mammary carcinoma cells by a
mammalian transient expression cloning.
Materials-UDP-GlcNAc was purchased from Sigma.
UDP-[C]GlcUA (285.2 mCi/mmol) was purchased from
DuPont NEN. UDP-GlcUA and MNNG (
)were purchased from Nakarai
Tesque, Kyoto, Japan. Sheep fixed erythrocytes were purchased from
Inter-Cell Technologies, Inc. Streptomyces hyaluronidase was
obtained from Seikagaku Corp., Tokyo, Japan. Superdex HR 10/30 column
was purchased from Pharmacia Biotech, Tokyo, Japan. A biotinylated
hyaluronan binding region (b-HABR), which specifically binds to
hyaluronan, was prepared from bovine nasal cartilage proteoglycan using
hyaluronan affinity chromatography according to the method of
Tengblad(13) .
Mutant cell lines deficient in hyaluronan biosynthesis were
isolated from FM3A HA1 cells mutagenized with MNNG. All the mutants
showed a considerably reduced level of hyaluronan production (less than
6 ng of HA/10 cells) compared with that of the wild-type
FM3A HA1 cell line (see Table 1). The genetic backgrounds of
these clones were analyzed by somatic cell fusion and resultant
complementation in hyaluronan biosynthesis. The clones were found to be
grouped into three classes (A, B, and C), and any combination of the
clones between the different classes complemented the hyaluronan
production (Table 1), which suggested that at least three genes
may contribute to hyaluronan biosynthesis and that the hyaluronan
synthesis would be restored if the normal gene is introduced into the
mutant cells. The typical clones representing class A and B maintained
significant levels of the hyaluronan synthase activity (45.2 ±
1.5 and 35.9 ± 2.3 pmol/h/mg of protein, respectively). By
contrast, the one clone representing class C almost lacked hyaluronan
synthase activity (Table 1) and was termed HAS
for subsequent study.
To clone a cDNA encoding a protein that
participates in hyaluronan synthase, a transient expression cloning
using HAS cells was adopted. One single clone that
directed the expression of hyaluronan in HAS
P cells
was finally isolated by sibling selection described under
``Experimental Procedures.'' We termed the cDNA isolated from
this clone HAS. HAS cDNA consisted of 2102 base pairs and a single long
open reading frame of 1749 base pairs (Fig. 1). Three methionine
codons are found within the first 35 codons of this reading frame, and
the most proximal one was assigned as the initiation codon, based on
Kozak's rules (21) for mammalian translation initiation.
This reading frame predicts a protein of 583 amino acids in length,
with a M
of 65,500. On the basis of Von
Heijne's(-3, -1) rule(22) , there is no
apparent NH
-terminal signal peptide sequence. Notable
stretches of hydrophobic amino acids exist in HAS protein (Fig. 1). The presence of those hydrophobic stretches in HAS
protein suggests that the protein may be associated with the membrane
via multiple membrane-spanning regions. The relative hydropathy of HAS
protein was further examined by plotting the hydrophobic index over the
entire length using the algorithm of Kyte and Doolittle(23) .
The analysis suggested the presence of the hydrophilic region between
the NH
-terminal hydrophobic stretches and the COOH-terminal
hydrophobic stretches (Fig. 2). In comparison with the
hydrophobicity profile of a bacterial hyaluronan synthase, HasA protein (24) , highly similar molecular arrangements were observed.
Mian (10) described previously that a 66-kDa protein may be a
constituent of the membrane-bound hyaluronan synthase complex partially
purified from the detergent-solubilized plasma membrane of cultured
human skin fibroblasts. Considering those, the structural
characteristics of HAS protein suggest that the protein may be either
the hyaluronan synthase itself or an essential component of the
synthase complex.
Figure 1: Nucleotide and deduced amino acid sequences of HAS. The open reading frame and full-length nucleotide sequences of clone HAS are shown. The initiation codon fits within the Kozak consensus sequence GCC(A/G)CCATGG in 7 out of 10 bases (indicated by small closed circles). The presumptive polyadenylation signal AATAAA is boxed. Hydrophobic stretches of amino acids (22-35, 52-67, 411-428, 436-457, 464-486, 501-519, 546-566) are underlined.
Figure 2: Hydropathic analysis of the deduced amino acid sequence of HAS. The hydrophobicity values were obtained according to the algorithm of Kyte and Doolittle(23) . Positive values represent increased hydrophobicity. The predicted membrane-associated domains are marked with open bars.
A computer search for proteins having identity to HAS protein was performed using the NBRF-PDB (release 45) data base. None of sequences identical to HAS protein was found in the protein data base. However, interestingly, a 557-amino acid overlap with 57.6% identity to HAS protein was observed with DG42 protein, the predicted product of an mRNA species that is rapidly accumulated and degraded during Xenopus laevis embryonic development(25) . DG42 protein had also been reported to be a protein homologous to HasA protein(9) . The search also showed significant homology between HAS protein and HasA protein (290-amino acid overlap with 33.1% identity). DeAngelis et al. (26) recently reported the presence of the conserved region of HasA protein among the various Group A Streptococci which is likely to be involved in the structure and/or function of the hyaluronan synthase. This region is also very similar in GlcNAc polymer synthases such as yeast chitin synthases and Rhizobium NodC(26) . Thus, we compared those regions among HAS, DG42, and HasA proteins using the GENETYX-MAC program (Fig. 3). The multiple sequence alignments of the regions showed that there is 76.7% identity between HAS and DG42, and 40.7% identity between HAS and HasA, respectively. The sequence conservation strongly supports that HAS gene product is greatly related to eukaryotic hyaluronan synthase. Although DG42 has recently been reported to synthesize oligosaccharide of GlcNAc but not hyaluronan in an in vitro transcription-translation system(27) , the in vitro translation system might have lost the one part of hyaluronan synthase activity that bears the GlcUA transferase activity, or it is also likely that other factor(s) might be necessary for the expression of the synthase activity.
Figure 3: Multiple sequence alignment of the HAS, DG42, and HasA proteins. Regions of approximately 150 amino acids were aligned using the GENETYX-MAC program. The amino acids displayed correspond to residues 242-391 for HAS, 240-389 for DG42, and 134-281 for HasA. Positions with two identical amino acids are denoted by asterisks, while those with three are denoted by boldface asterisks.
A transient expression of HAS
cDNA in HASP cells complemented the deficient matrix
deposition of hyaluronan (Fig. 4). Pretreatment of the cells
with Streptomyces hyaluronidase completely abolished the
formations of hyaluronan matrix surrounding the transfectants (Fig. 4D). After hyaluronidase digestion, the areas of
the hyaluronan matrix were identical to those observed in control
(transfectants with pcDNAI) (Fig. 4C). The
neo-synthesis of hyaluronan by HAS cDNA was confirmed using stable
transfectants expressing the HAS gene. The cell line
established from HAS
cells transfected with
pcDNA3-HAS synthesized and secreted hyaluronan at significantly higher
levels in the culture medium (Table 1). The control transfectants
with pcDNA3 vector produced low levels of hyaluronan. These results
again support that HAS protein takes part in the essential step of
hyaluronan biosynthesis. To obtain evidence for the possibility that
HAS cDNA encodes hyaluronan synthase, membrane fractions of those
stable transfectants were assayed for the hyaluronan synthase activity
as described under ``Experimental Procedures.'' The
significant activity was detected in the fractions from the
transfectants with HAS cDNA (Table 1).
Figure 4:
Visualization of HA matrices around
wild-type FM3A HA1 cells and transfectants. The fixed erythrocyte
exclusion assay was used to outline the hyaluronan matrix surrounding
cells. The hyaluronan matrix occupies the clear area (arrowheads) between the fixed erythrocytes and wild-type FM3A
HA1 cells or transfectants (pcDNAI-HAS). These photomicrographs were
taken on an Olympus IMT-2 inverted phase-contrast microscope at
200 magnification. A, wild-type FM3A HA1 cells; B,
transfectants (pcDNAI-HAS); C, transfectants (pcDNAI); D, transfectants (pcDNAI-HAS) treated with 1 turbidity
reducing unit/ml Streptomyces hyaluronidase for 1 h at 37
°C. The results depict the ability of plasmid pcDNAI-HAS, but not
pcDNAI alone, to direct hyaluronan matrix in the mutant HAS
cells.
Over all, our data demonstrate that the HAS gene product is responsible for the activity of the hyaluronan synthase and may correspond to synthase itself. Future studies on the activity of recombinant HAS protein will give the final conclusion.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D82964[GenBank].