Cloning of a mucin-desulfating sulfatase gene from Prevotella strain RS2 and its expression using a Bacteroides recombinant system

Abstract

A gene encoding the mucin-desulfating sulfatase in Prevotella strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into Escherichia coli expression vectors. However, cloning the gene into a Bacteroides expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.

FIG. 1

Restriction map of the 4.7-kb gDNA fragment from Prevotella strain RS2 cloned in pGEM-7zf(−). Arrows indicate the positions and directions of ORFs encoding the mucin-desulfating sulfatase (mdsA), the putative sulfatase posttranslational modification protein (mdsB), and a gene sequence tentatively identified as the N-terminal region of anthranilate phosphoribosyltransferase (apt). The cross-hatched region in mdsA shows the location of the 600-bp PCR product formed using primers TR4(F) and TR7(R). Nucleotide numbers refer to the 5,058 bp found after sequencing of the gDNA fragment. Restriction sites are indicated by the letters N (NcoI), Ps (PstI), Pv (PvuII), Sc (SacI), Sl (SalI), Sp (SphI), Su (Sau3AI), Xb (XbaI), and Xh (XhoI).

FIG. 2

Alignment of the deduced amino acid sequence of Prevotella strain RS2 mucin-desulfating sulfatase (MdsA-prev) with three other sulfatases: Yidj-esch, from the SWISS-PROT database, a putative E. coli sulfatase (f497 in reference 2); Gl6s-human, human N-acetylglucosamine-6-sulfatase (G6S in reference 21); and AtsA-kleb, K. pneumoniae arylsulfatase (14). Amino acids that occur in three of the four sequences are highlighted in reverse type, and a consensus sequence is included. Where the consensus sequence is highly conserved, the amino acids are underlined.

FIG. 3

Western blot analysis of recombinant and normal Prevotella strain RS2 mucin-desulfating sulfatases after SDS-PAGE. Samples analyzed included the periplasm of chondroitin sulfate-grown B. thetaiotaomicron containing plasmid pVGA-3 with the mdsA insert (7 μg of protein) (lane 1) and without the mdsA insert (7 μg of protein) (lane 2), periplasm of glucose-grown B. thetaiotaomicron containing plasmid pVGA-3 with the mdsA insert (7 μg of protein) (lane 3), periplasm of mucin-grown Prevotella strain RS2 (7 μg of protein) (lane 4), lysed spheroplasts of chondroitin sulfate-grown B. thetaiotaomicron containing plasmid pVGA-3 with the mdsA insert (16 μg of protein) (lane 5) and without the mdsA insert (23 μg of protein) (lane 6), and lysed spheroplasts of glucose-grown B. thetaiotaomicron containing plasmid pVGA-3 with the mdsA insert (9 μg of protein) (lane 7). Molecular size standards are shown on the left.