Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX.

Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysaccharidosis IX, was recently described. This patient had a surprisingly mild clinical phenotype, including notable periarticular soft tissue masses, mild short stature, an absence of neurological or visceral involvement, and histological and ultrastructural evidence of a lysosomal storage disease. To determine the molecular basis of mucopolysaccharidosis IX, we analyzed two candidate genes tandemly distributed on human chromosome 3p21.3 and encoding proteins with homology to a sperm protein with hyaluronidase activity. These genes, HYAL1 and HYAL2, encode two distinct lysosomal hyaluronidases with different substrate specificities. We identified two mutations in the HYAL1 alleles of the patient, a 1412G --> A mutation that introduces a nonconservative amino acid substitution (Glu268Lys) in a putative active site residue and a complex intragenic rearrangement, 1361del37ins14, that results in a premature termination codon. We further show that these two hyaluronidase genes, as well as a third recently discovered adjacent hyaluronidase gene, HYAL3, have markedly different tissue expression patterns, consistent with differing roles in HA metabolism. These data provide an explanation for the unexpectedly mild phenotype in mucopolysaccharidosis IX and predict the existence of other hyaluronidase deficiency disorders.

Structural and functional characterization of Streptomyces plicatus beta-N-acetylhexosaminidase by comparative molecular modeling and site-directed mutagenesis.

We have sequenced the Streptomyces plicatus beta-N-acetylhexosaminidase (SpHex) gene and identified the encoded protein as a member of family 20 glycosyl hydrolases. This family includes human beta-N-acetylhexosaminidases whose deficiency results in various forms of GM2 gangliosidosis. Based upon the x-ray structure of Serratia marcescens chitobiase (SmChb), we generated a three-dimensional model of SpHex by comparative molecular modeling. The overall structure of the enzyme is very similar to homology modeling-derived structures of human beta-N-acetylhexosaminidases, with differences being confined mainly to loop regions. From previous studies of the human enzymes, sequence alignments of family 20 enzymes, and analysis of the SmChb x-ray structure, we selected and mutated putative SpHex active site residues. Arg162 --> His mutation increased Km 40-fold and reduced Vmax 5-fold, providing the first biochemical evidence for this conserved Arg residue (Arg178 in human beta-N-acetylhexosaminidase A (HexA) and Arg349 in SmChb) as a substrate-binding residue in a family 20 enzyme, a finding consistent with our three-dimensional model of SpHex. Glu314 --> Gln reduced Vmax 296-fold, reduced Km 7-fold, and altered the pH profile, consistent with it being the catalytic acid residue as suggested by our model and other studies. Asp246 --> Asn reduced Vmax 2-fold and increased Km only 1.2-fold, suggesting that Asp246 may play a lesser role in the catalytic mechanism of this enzyme. Taken together with the x-ray structure of SmChb, these studies suggest a common catalytic mechanism for family 20 glycosyl hydrolases.

Characterization of the murine beta-hexosaminidase (HEXB) gene.

The murine HEXB gene, encoding the beta-subunit of the lysosomal hydrolase, beta-hexosaminidase, was isolated from a mouse cosmid library as a single cosmid clone. The entire gene spans 22 kb, considerably less than the 40 kb spanned by its human counterpart. It is highly homologous to the human gene. The 14 intron-exon junctions are entirely conserved, although the intron sequences diverge rapidly. Upstream of the coding region, a 1.3 kb segment was sequenced and shown to function as a promoter when fused with a reporter gene and expressed in monkey COS-7 cells. A short sequence (100 bp), near the start of the coding region, exhibits strong homology to the human HEXB promoter. Analysis of the tissue distribution of the HEXB mRNA in 129/Sv male mice revealed up to 28-fold tissue-specific variations in transcript levels. The kidney and the epididymis had the highest mRNA levels consistent with past surveys of enzyme activity.