Lack of sialic acid in synovial fluid fibronectin

Abstract

not received ~ynovial fluid F~~ronectin Sialic acid deficiency 1, INTRODUCTION fibronectin by proteolytic enzymes present in synovial fluid [lo]. Fibronectins are high h4, glycoproteins present in an insoluble form in int~rstiti~ matrices, basement membranes, newly formed connective tissue, differentiating cartilage and bone, and rheumatoid synovial membrane. Fibronectins are also present, in a soluble form, in blood plasma and other body fluids including amniotic, synovial, cerebrospinal and pleural fluids, urine and milk (for reviews on distribution, structure and biological functions of fibronectin see [l-6]). Here we demonstrate that charge differences between plasma and synovial fluid fibronectin are not due to proteolytic cleavage but to lack of sialic acid in synovial fluid fibronectin. 2. MATERIALS AND METHODS The functions attributed to fibronectin such as cell adhesion to substrata, cell spreading, opsonization of bacteria and other particulate matter, ability to induce a more normal phenotype in transformed cells, wound healing and chemotaxis are all related to its affinity to cell surface and a large number of different macromolecules (see reviews above). In previous studies fibron~ctin has been reported to be present in synovial fluid from patients with various rheumatic diseases and exceed those in plasma samples from the same individual, suggesting local synthesis [7-lo]. Recently it was reported that synovial fluid ~bronectin from rheumatoid patients has a different molecular charge with respect to plasma fibronectin [lo]. This different molecular charge has been attributed to proteolytic cleavage of Synovial fluid and blood were supplemented with EDTA (final cont. 0.590, w/v). Synovial fluids were incubated with hyaluronidase (final cont. 5 pg/ml) at 37°C for 1 h. Samples were centrifuged at 7000 x g for 10 min at 20°C. Sodium azide at a final cont. of 0.1% (w/v) and 5 kunits/ml of aprotinin (Sigma, St. Louis, MO) were added. In experiments comparing plasma and synovial fluid fibronectin the plasma was also incubated with hyaluronidase (5 @g/ml). Human plasma and synovial fluid fibronectin were purified as in [ 11,121. Sodium dodecylsulphatepolyacrylamide gel electrophoresis (SDS-PAGE) was done as in [ 131. Separation of plasma and synovial fluid proteins according to their net molecular charge was carried out either on 5% polyacrylamide gels as in [f4] or on 2% agarose in high resolution buffer (Gelman Sciences, Ann Arbor, MI). * To whom reprint requests should be addressed plasma and synovial fluid proteins were transferred from polyacrylamide gels to nitrocellulose sheets as in [ 151. Fibronectin was located on nitrocellulose sheets using monospecific Published by Elsevier Science Publishers B. V, 00145793/84/$3.00