Identification and Action Patterns of Two Chondroitin Sulfate Sulfatases From a Marine Bacterium Photobacterium sp. QA16

Abstract

Chondroitin sulfate (CS)/dermatan sulfate (DS) is a kind of sulfated polyanionic, linear polysaccharide belonging to glycosaminoglycan. CS/DS sulfatases, which specifically hydrolyze sulfate groups from CS/DS oligo-/polysaccharides, are potential tools for structural and functional studies of CD/DS. However, only a few sulfatases have been reported and characterized in detail to date. In this study, two CS/DS sulfatases, PB_3262 and PB_3285, were identified from the marine bacterium Photobacterium sp. QA16 and their action patterns were studied in detail. PB_3262 was characterized as a novel 4-O-endosulfatase that can effectively and specifically hydrolyze the 4-O-sulfate group of disaccharide GlcUAβ1–3GalNAc(4-O-sulfate) but not GlcUAβ1–3GalNAc(4,6-O-sulfate) and IdoUAα1–3GalNAc(4-O-sulfate) in CS/DS oligo-/polysaccharides, which is very different from the identified 4-O-endosulfatases in the substrate profile. In contrast, PB_3285 specifically hydrolyzes the 6-O-sulfate groups of GalNAc(6-O-sulfate) residues located at the reducing ends of the CS chains and is the first recombinantly expressed 6-O-exosulfatase to effectively act on CS oligosaccharides.

Cloning and Characterization of a Novel N-Acetyl-D-galactosamine-4-O-sulfate Sulfatase, SulA1, from a Marine Arthrobacter Strain

“…This speculation was supported by the characterization of ChoA1 as a chondroitin AC lyase [ 23 ], exhibiting activity towards CS-4S and CS-6S. Additionally, the amino acid sequence of SulA2 shares high topology conservation with the exolytic CS N -acetyl-galactosamine-6- O -sulfatase [ 7 ], with 96% coverage, which could be indicative for functional conservation. No activity was detected for either heparin or κ-carrageenan-derived saccharides, nor for the fucose-containing sulfated polysaccharides (FCSPs) from brown seaweed.…”

“…Notably, the conserved sulfatase Type 1 signature motif C/SXPXR was identified in SulA1 as well as in all the aligned homologous sequences, in line with the previous findings in the literature [ 8 , 9 , 30 ]. Notably, characterized CS/DS sulfatases [ 2 , 7 , 13 ] showed less than 30% identity compared to SulA1 in BLASTp analysis on NCBI, highlighting the novelty of SulA1 and the challenges in predicting substrate specificity based on its structure.…”

“…CS/DS-sulfatases have been further categorized based on their substrate specificity, namely Δ 4,5 hexuronate-2- O -sulfatase, N -acetylgalactosamine-4- O -sulfatase (GalNAc-4- O -sulfatase), and N -acetylgalactosamine-6- O -sulfatase (GalNAc-6- O -sulfatase). These sulfatases target specific sulfate groups within CS/DS molecules, enabling the modification and degradation of these biomolecules [ 6 , 7 ]. However, despite the discovery of new enzymes and a large amount of genomic data, sulfatases remain, to a large extent, underexplored concerning substrate specificity and kinetic properties [ 8 , 9 ].…”

“…However, despite the discovery of new enzymes and a large amount of genomic data, sulfatases remain, to a large extent, underexplored concerning substrate specificity and kinetic properties [ 8 , 9 ]. So far, only three Δ 4,5 hexuronate-2- O -sulfatase [ 10 , 11 , 12 ], three endo-type and two exo-type GalNAc-4- O -sulfatases [ 2 , 7 , 12 , 13 , 14 ], and three exo-type GalNAc-6- O -sulfatases [ 7 , 12 , 14 ], have been identified and characterized.…”

Data on cloning, expression and biochemical characteristics of a chondroitin sulfate/dermatan sulfate 4-O-endosulfatase

“…The competent cells E. coli BL21 (DE3) and Fast Mutagenesis Kit V2 were purchased from Vazyme (Nanjing, China). The sulfated CS disaccharides with various sulfation patterns, including Δ 4,5 HexUA1–3GalNAc(4S) (ΔA), Δ 4,5 HexUA1–3GalNAc(6S) (ΔC), Δ 4,5 HexUA(2S)1–3GalNAc(6S) (ΔD) and Δ 4,5 HexUA1–3GalNAc(4S,6S) (ΔE), were prepared by completely digesting several CS polysaccharides treated with CSase ABC, and separated by anion exchange High Performance Liquid Chromatography (HPLC) using a YMC-Pack PA-G column (YMC, Japan) [3] .…”