A novel mechanism for desulfation of mucin: identification and cloning of a mucin-desulfating glycosidase (sulfoglycosidase) from Prevotella strain RS2

Abstract

A novel enzyme which may be important in mucin degradation has been discovered in the mucin-utilizing anaerobe Prevotella strain RS2. This enzyme cleaves terminal 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sulfate (6-SO3-GlcNAc) residues from sulfomucin and from the model substrate 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. The existence of this mucin-desulfating glycosidase (sulfoglycosidase) suggests an alternative mechanism by which this bacterium may desulfate sulfomucins, by glycosidic removal of a sulfated sugar from mucin oligosaccharide chains. Previously, mucin desulfation was thought to take place by the action of a specific desulfating enzyme, which then allowed glycosidases to remove desulfated sugar. Sulfate removal from sulfomucins is thought to be a rate-limiting step in mucin degradation by bacteria in the regions of the digestive tract with a significant bacterial flora. The sulfoglycosidase was induced by growth of the Prevotella strain on mucin and was purified 284-fold from periplasmic extracts. Tryptic digestion and sequencing of peptides from the 100-kDa protein enabled the sulfoglycosidase gene to be cloned and sequenced. Active recombinant enzyme was made in an Escherichia coli expression system. The sulfoglycosidase shows sequence similarity to hexosaminidases. The only other enzyme that has been shown to remove 6-SO3-GlcNAc from glycoside substrates is the human lysosomal enzyme beta-N-acetylhexosaminidase A, point mutations in which cause the inheritable, lysosomal storage disorder Tay-Sachs disease. The human enzyme removes GlcNAc from glycoside substrates also, in contrast to the Prevotella enzyme, which acts on a nonsulfated substrate at a rate that is only 1% of the rate observed with a sulfated substrate.

FIG. 1.

SDS-PAGE of sulfoglycosidase. (a) SGL purified from Prevotella strain RS2. Lane 1, hydroxyapatite column fraction A (2.9 μg of protein) (see Table 1); lane 2, hydroxyapatite column fraction B (2.0 μg); right lane, protein molecular weight standard (0.5 μg). (b) Recombinant SGL. Left lane, protein molecular weight standard (5 μg); lane 1, supernatant from sonicated E. coli, containing recombinant SGL; lane 2, enzyme eluted from Ni-NTA column; lane 3, enzyme eluted from hydroxyapatite chromatography column; lane 4, enzyme eluted by Q Sepharose chromatography. The gels were stained with Coomassie brilliant blue.

FIG. 2.

Alignment of the deduced amino acid sequence of sulfoglycosidase from Prevotella strain RS2 with homologous sequences. Prevotella RS2, sulfoglycosidase from Prevotella strain RS2; B. thetaiotaomicron, β-hexosaminidase precursor from B. thetaiotaomicron; Homo sapiens, β-N-acetylhexosaminidase subunit A from H. sapiens. Alignment was carried out by using Clustal W (1.82) multiple-sequence alignment. Fully conserved residues (asterisks) and residues with one conservative replacement (colons) are highlighted.

FIG. 3.

Paper chromatography of product from digestion of pig gastric mucin by recombinant sulfoglycosidase. PGM_t was incubated with recombinant SGL as described in the text. The product was subjected to ascending paper chromatography performed with solvent system A. Lane 1, PGM_t plus buffer (negative control, no SGL); lane 2, PGM_t incubated with SGL and buffer; lane 3, buffer (negative control, no PGM_t, no SGL); lane 4, SGL plus buffer (negative control, no PGM_t); lane 5, galactose standard (0.1 μmol); lane 6, 6-SO₃-Gal standard (0.1 μmol); lane 7, GlcNAc standard (0.1 μmol); lane 8, 6-SO₃-GlcNAc standard (0.1 μmol). The arrowheads indicate the sample loading line and the solvent front. The chromatogram was stained by using the dip method of Trevelyan et al (23). The spot at an R_f of 0.62 occurs in all lanes containing material filtered through a dialysis membrane (cutoff, 10 kDa) (lanes 1 to 4) and is likely to be glycerin or a sulfur compound eluted from the membrane.