Biotinylated hyaluronan: a versatile and highly sensitive probe capable of detecting nanogram levels of hyaluronan binding proteins (hyaladherins) on electroblots by a novel affinity detection procedure.
Hyaluronan influences cellular proliferation and migration in developing, regenerating and remodelling tissues and in tissues undergoing malignant tumour-cell invasion. The widespread occurrence of hyaluronan-binding proteins indicates that the recognition of hyaluronan is important to tissue organisation and the control of cellular behaviour. A number of extracellular matrix and cellular proteins, which have been termed the hyaladherins, have specific affinities for hyaluronan. These include cartilage link-protein, hyaluronectin, neurocan, versican and aggrecan, which all bind to HA within the extracellular matrix. Cellular receptors for hyaluronan such as CD44 and RHAMM (receptor for hyaluronate-mediated motility) have also been identified. In the present study biotinylated hyaluronan (bHA) was prepared by reacting adipic dihydrazide with a 170 kDa hyaluronan sample using the bifunctional reagent 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide. The resultant free amine moeity of the hydrazido-hyaluronan was then reacted with biotin succinimidyl ester (sulfo-NHS-biotin) to prepare the bHA. After 4-20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotting to nitrocellulose membranes, bHA and avidin alkaline phosphatase conjugate could be used in conjunction with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate substrates to specifically visualise with high sensitivity (> or = 2 ng), bovine nasal cartilage link-protein, aggrecan hyaluronan binding region, and human fibroblast hyaluronan receptors such as CD-44. Conventional Western blotting using specific monoclonal antibodies to these proteins was also used to confirm the identities of these proteins.
Melrose
J
J
Raymond Purves Bone and Joint Research Laboratories, The Royal North Shore Hospital of Sydney and The University of Sydney, St. Leonards, Australia.
Numata
Y
Y
Ghosh
P
P
D053674:Aggrecans; D000818:Animals; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry*; D015153:Blotting, Western; D002417:Cattle; D002847:Chromatography, Agarose; D003101:Collodion; D004563:Electrochemistry; D016326:Extracellular Matrix Proteins; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D018960:Hyaluronan Receptors / Q000032:analysis*; D006820:Hyaluronic Acid / Q000737:chemistry*; D037181:Lectins, C-Type; D008832:Microchemistry; D015335:Molecular Probes; D011506:Proteins / Q000032:analysis*; D011509:Proteoglycans / Q000032:analysis*; D012680:Sensitivity and Specificity; D000099249:Proteoglycan Link Protein
D053674:Aggrecans; D001710:Biotin; D003101:Collodion; D016326:Extracellular Matrix Proteins; D018960:Hyaluronan Receptors; D006820:Hyaluronic Acid; D037181:Lectins, C-Type; D015335:Molecular Probes; D011506:Proteins; D011509:Proteoglycans; D000099249:Proteoglycan Link Protein