Monoclonal antibody MT2 identifies the urodele alpha 1 chain of type XII collagen, a developmentally regulated extracellular matrix protein in regenerating newt limbs.
We previously described the upregulation of the MT2 antigen during urodele limb regeneration and characterized the MT2 antigen as a 310- to 325-kDa chondroitin-sulfated glycoprotein with a core protein of 285-300 kDa. In this study, we screened a newt blastema cDNA library using monoclonal antibody (mAb) MT2 and obtained a 1-kb cDNA fragment, designated Isolate (IS)-1. Subsequent screening of the same library using IS-1 cDNA as a probe provided IS-2, a 2.8-kb cDNA. IS-2 overlaps IS-1 at its 5' end, is highly homologous to a portion of the alpha 1 chain of 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 from cDNA IS-2 demonstrates invariable tryptophan, leucine, threonine, and tyrosine residues that are highly conserved among all the fibronectin type III domains within IS-2 and between corresponding sequences of IS-2 and chicken alpha 1[XII]. A Northern blot showed a 10-kb band that 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 malE gene of Escherichia coli, which encodes maltose-binding protein (MBP). The isopropyl beta-D-thiogalactoside-induced fusion protein had the expected molecular weight and reacted to both mAb MT2 and rabbit anti-MBP serum. We conclude that mAb MT2 identifies the urodele alpha 1[XII]. The expression pattern of the type XII collagen gene in newt limb 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 transcriptionally active. At mid-bud and late-bud blastema stages, wound epithelium expression had decreased, whereas the mesenchyme remained strongly active in transcription and showed a tendency toward distal regionalization. Condensing cartilage showed no signal. Finally, at the late digit stage, hybridization became largely restricted to the perichondrium. The in situ results suggest a developmental role for type XII collagen in regeneration.
Wei
Y
Y
Neuroscience Graduate Studies Program, Ohio State University, Columbus 43210, USA.
Yang
E V
EV
Klatt
K P
KP
Tassava
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D002645:Chickens; D003094:Collagen / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005109:Extracellular Matrix / Q000378:metabolism*; D005121:Extremities / Q000473:pathology / Q000502:physiology*; D008969:Molecular Sequence Data; D012038:Regeneration; D016415:Sequence Alignment; D014562:Caudata / Q000502:physiology*
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D003094:Collagen