MONOCLONAL-ANTIBODY MT2 IDENTIFIES THE URODELE ALPHA-1 CHAIN OF TYPE-XII COLLAGEN, A DEVELOPMENTALLY-REGULATED EXTRACELLULAR-MATRIX PROTEININ REGENERATING NEWT LIMBS

We previously described the upregulation of the MT2 antigen during uro dele limb regeneration and characterized the MT2 antigen as a 310- to 325-kDa chondroitin-sulfated glycoprotein with a core protein of 285-3 00 kDa. In this study, we screened a newt blastema cDNA library using monoclonal antibody (mAb) MT2 and obtained a l-kb cDNA fragment, desig nated Isolate (IS)-1, Subsequent screening of the same library using I S-1 cDNA as a probe provided IS-2, a 2.8-kb cDNA. IS-2 overlaps IS-1 a t its 5' end, is highly homologous to a portion of the alpha 1 chain o f the chicken type XII collagen cDNA (alpha 1[XII]), and spans a third of the chicken alpha 1[XII] cDNA, from the last 62 amino acids of the second A domain of von Willebrand factor to the first two repeats of the fourth fibronectin type III domain. The peptide sequence deduced f rom cDNA IS-2 demonstrates invariable tryptophan, leucine, threonine, and tyrosine residues that are highly conserved among all the fibronec tin type III domains within IS-2 and between corresponding sequences o f IS-2 and chicken alpha 1[XII]. A Northern blot showed a 10-kb band t hat corresponds to the size of the chicken alpha 1[XII] mRNA, A fusion gene was constructed by inserting the IS-2 cDNA downstream from the m alE gene of Escherichia coil, which encodes maltose-binding protein (M BP). The isopropyl beta-D-thiogalactoside-induced fusion protein had t he expected molecular weight and reacted to both mAb MT2 and rabbit an ti-MBP serum, We conclude that mAb MT2 identifies the urodele alpha 1[ XII]. The expression pattern of the type XII collagen gene in newt lim b regenerates was examined by in situ hybridization. Type XII collagen transcripts first appeared at 3 days after amputation in cells of the basal layer of the wound epithelium. At Day 10, both the basal wound epithelial cells and the distal mesenchyme cells were highly transcrip tionally active, At mid-bud and late-bud blastema stages, wound epithe lium expression had decreased, whereas the mesenchyme remained strongl y active in transcription and showed a tendency toward distal regional ization, Condensing cartilage showed no signal. Finally, at the late d igit stage, hybridization became largely restricted to the perichondri um, The in situ results suggest a developmental role for type XII coll agen in regeneration. (C) 1995 Academic Press, Inc.