Spicer AP and McDonald JA (1998), Characterization and molecular evolution of a v...

XB-ART-15560

J Biol Chem 1998 Jan 23;2734:1923-32.

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Characterization and molecular evolution of a vertebrate hyaluronan synthase gene family.

Spicer AP , McDonald JA .

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The three mammalian hyaluronan synthase (HAS) genes and the related Xenopus laevis gene, DG42, belong to a larger evolutionarily conserved vertebrate HAS gene family. We have characterized additional vertebrate HAS genes from chicken (chas2 and chas3) and Xenopus (xhas2, xhas3, and a unique Xenopus HAS-related sequence, xHAS-rs). Genomic structure analyses demonstrated that all vertebrate HAS genes share at least one exon-intron boundary, suggesting that they evolved from a common ancestral gene. Furthermore, the Has2 and Has3 genes are identical in structure, suggesting that they arose by a gene duplication event early in vertebrate evolution. Significantly, similarities in the genomic structures of the mouse Has1 and Xenopus DG42 genes strongly suggest that they are orthologues. Northern analyses revealed a similar temporal expression pattern of HAS genes in developing mouse and Xenopus embryos. Expression of mouse Has2, Has3, and Xenopus Has1 (DG42) led to hyaluronan biosynthesis in transfected mammalian cells. However, only mouse Has2 and Has3 expressing cells formed significant hyaluronan-dependent pericellular coats in culture, implying both functional similarities and differences among vertebrate HAS enzymes. We propose that vertebrate hyaluronan biosynthesis is regulated by a comparatively ancient gene family that has arisen by sequential gene duplication and divergence.

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Species referenced: Xenopus laevis
Genes referenced: actl6a cela1 has-rs has1 has2 has3

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	FIG. 1. Multiple amino acid sequence alignment of vertebrate HAS enzymes obtained from degenerate PCR products. Amino acid sequences are aligned with reference to mouse Has2 (mHas2) amino acids 271â461 (16). Dashes indicate sequence identity; spaces indicate gaps that have been introduced to maximize the alignment. Significantly, sequence differences among vertebrate HAS enzymes have been conserved between species. Thus, sequences could be classified as Has2, Has3, etc. m, mouse; h, human; c, chicken (G. gallus); x, X. laevis; z, zebrafish (D. rerio). The zebrafish Has2 (zHas2) sequence was added from previously published data (27).
	FIG. 2. Nucleotide and predicted amino acid sequence of X. laevis HAS-related sequence, xHAS-rs. The open reading frame is capitalized. 59- and 39-untranslated regions are indicated in lowercase. The predicted start codon is underlined as is the predicted stop codon, an in-frame stop codon within the 59-untranslated region, and the consensus polyadenylation signal.
	FIG. 3. Comparison of vertebrate HAS enzymes and HAS-related sequence. A, multiple amino acid sequence alignment between known vertebrate HAS enzymes and HAS-related sequences (HAS-rs). Boxed residues indicate positions within the proteins where at least 3 out of 5 residues are identical. Dashes indicate gaps that have been introduced to maximize the alignment. m, mouse; x, X. laevis. B, amino acid sequence alignment of three regions of the vertebrate HAS proteins with equivalent regions of related b-glycosaminyltransferases. m, mouse; x, X. laevis; HasA, S. pyogenes hyaluronan synthase; NodC, Rhizobium meliloti nodulation factor C; celA1, Gossypium hirsutum (cotton) putative cellulose synthase A1; Chs2, Saccharomyces cerevisiae chitin synthase 2. Highly conserved residues are boxed. Site-directed mutagenesis of the Chs2 residues indicated by the asterisks resulted in loss of enzymatic activity (39).
	FIG. 4. Genomic structure of vertebrate HAS and HAS-rs genes. A, restriction maps of the mouse Has genes drawn to scale with the X. laevis has1 (DG42) and HAS-related sequence (xHAS-rs) genes. Exons are indicated by boxes. Open boxes indicate 59- and 39-untranslated regions; filled boxes indicate the open reading frame. The respective locations of start codons (ATG) and stop codons (TAA and TGA) are indicated. Restriction sites for the enzymes, BamHI (B), SacII (C), EcoRI (E), HindIII (H), KpnI (K), HpaI (P), and SacI (S) are shown. B, alignment of vertebrate HAS and HAS-rs gene exons based upon exon-intron boundaries. Exons are represented by numbered boxes. The position of exon-intron boundaries is represented by gaps between each exon. Open boxes indicate 59- and 39-untranslated regions; filled boxes indicate the open reading frame. The location of the start codon (ATG) is indicated for each gene. The position of AUUUA elements within 39-untranslated regions is noted. Based upon these alignments and the results from Table I, the HAS genes could be grouped into two classes: xhas1 (DG42), mouse Has1, and xHAS-rs comprising one class, and mouse Has2 and mouse Has3 the other.
	FIG. 5. Northern analysis of vertebrate HAS gene expression. A, expression of HAS and HAS-rs genes in the developing mouse and Xenopus embryos. HAS-specific radiolabeled probes were sequentially hybridized to Northern blots of staged embryonic RNAs. Xenopus developmental stages were as follows: stage 1, fertilized egg; stage 8, mid-blastula transition; stage 12, gastrula; stage 19, neurula; stage 25, early tadpole. The mouse blot was hybridized with a b-actin probe to determine variation in sample loading. The Xenopus blot was stained with methylene blue prior to hybridization to detect ribosomal RNAs (rRNA) to determine efficiency of transfer and variation in sample loading. All probes were labeled to similar specific activities, and membranes were exposed to autoradiographic film for identical lengths of time; mouse poly(A)1 RNA blot, 16 h at 280 Â°C; Xenopus total RNA blot, 3 days at 280 Â°C. B, expression of HAS genes in a panel of human adult tissues. Membranes were sequentially hybridized with partial cDNA probes for the respective human HAS genes. To determine variation in sample loading membranes were hybridized with a human b-actin probe. Membranes were exposed to autoradiographic film for an average of 5 days at 280 Â°C. Sk., skeletal; Sm., small. PBL, peripheral blood leukocyte.
	FIG. 6. Functional analysis of vertebrate HAS enzymes in vitro. Top, COS-1 cells were transfected with the respective HAS expression vectors (panels bâh) or with a control (empty) vector (panel a), and HA coat assays were performed as described previously (16). Only COS-1 cells transfected with mouse Has2 (panel e) or mouse Has3 (panel g) expression vectors generated significant pericellular coats that were specifically destroyed by treatment with S. hyaluronlyticus hyaluronidase (panels f and h). Identical results were observed in a second mammalian cell line, human 293 cells (data not shown). Bottom, agarose gel electrophoresis of radiolabeled hyaluronan synthesized in vitro by crude cell membranes prepared from COS-1 cells expressing vertebrate HAS genes. HA synthase assays were divided into two tubes and incubated overnight at 60 Â°C in the presence (1) or absence (2) of 1 TRU of S. hyalurolyticus hyaluronidase (Hase) at approximately pH 5.0. Samples were analyzed by agarose gel electrophoresis as described under âExperimental Procedures.â
	FIG. 7. Characterization of HAS-reactive antisera by immunoblotting. Crude membrane preparations from COS-1 cells transfected with vertebrate HAS expression vectors or pCIneo control vector were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting as described under âExperimental Procedures.â Antisera were raised against mouse Has2-derived synthetic peptides or against an N-terminal portion of Xenopus Has1 (DG42). One antiserum, MC287, raised against mouse Has2 peptide 2, cross-reacted with Xenopus Has1 (DG42), mouse Has1, mouse Has2, and mouse Has3 (left panel). A second antiserum, MC285, raised against mouse Has2 peptide 1, only detected mouse Has2 by immunoblotting (middle panel). The antixHas1 (DG42) N-terminal antiserum was completely specific for Xenopus Has1 (DG42) and failed to detect mouse HAS proteins even with prolonged exposures (right panel), suggesting that this antiserum is not suitable for detecting HAS proteins other than xHas1 (DG42) by immunochemistry.