Monoclonal antibody MT2 identifies an extracellular matrix glycoprotein that is co-localized with tenascin during adult newt limb regeneration

Using immunohistochemical techniques and mAb MT2, we describe here a novel extracellular matrix (ECM) molecule that is developmentally regulated during limb regeneration in adult newts. The MT2 antigen appears during preblastema stages, is most abundant during blastema stages, and persists, near undifferentiat-ed cells, until digit stages. The MT2 antigen is located in an acellular layer under the wound epithelium and throughout the ECM of the undifferentiated mesen-chyme as a thick, cord-like component. In unamputated limbs mAb MT2 reactivity is restricted to tendons, myo-tendinous junctions, periosteum and to a layer of material beneath the epidermis. In both unamputated limbs and regenerating limbs, the reactivity to mAb MT2 colo-calizes closely with urodele tenascin. Immunoblot analysis of blastema extracts showed that the unreduced form of the MT2 antigen is a large, polydispersed protein of approximately the same size as tenascin. However, based upon (a) molecular weights of reduced subunits, (b) competition experiments on tissue sections, and (c) analysis of molecules immunopreci-pitated by mAb MT2, we conclude that the MT2 substance is unrelated biochemically to tenascin. The results from immunoblots, enzyme digestions and DEAE-Se-phacell binding studies suggest that the unreduced MT2 antigen is a large protein composed of subunits which are connected by disulfide bonds. Reduction of the MT2 antigen results in three components recognized by mAb MT2. The largest of these reduced components is a chondroitin sulfate-like glycoprotein with a molecular weight (Mr) of 310–325 × 10³. A second component (Mr, 285–300 × 10³) is the core protein of the 310–325 × 10³ glycoprotein. The smallest reduced component, which may be a degradation product of the two larger components, has a Mr of 250–275 × 10³.