Biosensor analysis of the molecular interactions of pentosan polysulfate and of sulfated glycosaminoglycans with immobilized elastase, hyaluronidase and lysozyme using surface plasmon resonance (SPR) technology.
Pentosan polysulfate (NaPPS) and chondroitin sulfates (ChSs) have recently been shown to exhibit both symptom and disease modifying activities in osteoarthritis (OA), but their respective mechanisms of action are still the subject of conjecture. Excessive catabolism of joint articular cartilage is considered to be responsible for the initiation and progression of OA but the abilities of these drugs to mitigate this process has received only limited attention. Human neutrophil elastase (HNE) is a proteinase, which can degrade the collagens and proteoglycans (PGs) of the cartilage directly or indirectly by activating latent matrix metalloproteinases. Hyaluronidase (HAase) is an endoglycosidase, which degrades glycosaminoglycans including hyaluronan, which provides the aggregating component of the PG aggrecan complex. In the present study the molecular interactions between the NaPPS, ChSs and some other sulfated polysaccharides with immobilized HNE, HAase or lysozyme (a cationic protein implicated in PG metabolism) were studied using a SPR biosensor device-BIAcore2000. The above three enzymes were covalently immobilized to a biosensor chip CM5 separately using amine coupling. The binding affinity of each sulfated polysaccharide and the kinetics of NaPPS over the concentration range of 0.3-5.0 microg/ml were determined. The inhibition of HNE by the sulfated polysaccharides as determined using the synthetic substrate succinyl-Ala-Ala-Val-nitroanilide (SAAVNA) in a functional assay was compared with their respective binding affinities for this proteinase using the BIAcore system. The results obtained with the two independent techniques showed good correlation and indicated that the degree and ring positions of oligosaccharide sulfation were major determinants of enzyme inhibitory activity. The observed difference in order of binding affinities of the drugs to the immobilized HNE, HAase and lysozyme suggests a conformational relationship, in addition to the charge interactions between the sulfate esters of the polysaccharides and the cationic amino acids of the enzymes. Significantly, the SPR biosensor technology demonstrated that small differences among sulfated polysaccharides, even subtle variations among different NaPPS batches, could be readily detected. The SPR technology therefore offers not only a sensitive and reproducible method for ranking noncompetitive enzyme inhibitors for drug discovery but a rapid and quantitative bioassay for monitoring batch consistency of manufacture.
Shen
Bojiang
B
Institute of Bone and Joint Research, Level 5, University Clinic, Royal North Shore Hospital of Sydney, St. Leonards, NSW 2065, Australia.
Shimmon
Susan
S
Smith
Margaret M
MM
Ghosh
Peter
P
D015374:Biosensing Techniques; D002240:Carbohydrate Sequence; D004800:Enzymes, Immobilized; D006025:Glycosaminoglycans / Q000737:chemistry / Q000494:pharmacology*; D006821:Hyaluronoglucosaminidase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007202:Indicators and Reagents; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D009113:Muramidase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010426:Pentosan Sulfuric Polyester / Q000737:chemistry / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
D004800:Enzymes, Immobilized; D006025:Glycosaminoglycans; D007202:Indicators and Reagents; D008024:Ligands; D010426:Pentosan Sulfuric Polyester; D009113:Muramidase; D006821:Hyaluronoglucosaminidase; D010196:Pancreatic Elastase