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Partial characterization of a low molecular weight human collagen that undergoes alternative splicing
Abstract:
A cDNA library prepared from RNA isolated from a cultured human tumor cell line, HT-1080, was screened with a mouse cDNA clone coding for part of the -Gly-Xaa-Yaa- domain of the alpha 2(IV) collagen chain. Four overlapping cDNA clones were characterized that coded for a low molecular weight human collagen. The cDNA clones did not, however, code for the short-chain collagens, types IX and X. The amino acid sequences derived from the clones resembled type IV collagen in that there were short interruptions in the repeating -Gly-Xaa-Yaa- sequence. The noncollagenous, carboxyl-terminal domain was, however, much shorter and contained only 18 amino acid residues. Interestingly, one of the cDNA clones contained an additional 36 nucleotides not found in an overlapping clone. The 36 nucleotides encoded four -Gly-Xaa-Yaa- repeats without changing the reading frame. Nuclease S1 mapping demonstrated that the difference between the clones was due to existence of two different mRNAs. A synthetic 24-residue peptide corresponding to the last two -Gly-Xaa-Yaa- triplets and the entire carboxyl-terminal domain was used to generate polyclonal antibodies. Electrophoretic transfer blot analysis of HT-1080 cells and normal human skin fibroblasts identified two polypeptides, Mr 67,000 and Mr 62,000, that were sensitive to bacterial collagenase.
Insights
Researchers identified a novel low molecular weight human collagen from tumor cells. This collagen exhibits structural similarities to type IV collagen but has a shorter C-terminal domain and exists in two mRNA forms.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Type IV collagen is a major component of basement membranes.
- Understanding collagen diversity is crucial for cell biology and disease research.
Purpose of the Study:
- To characterize a novel low molecular weight human collagen.
- To investigate the structural and genetic basis of this collagen.
Main Methods:
- Screening of a human tumor cell line (HT-1080) cDNA library.
- Characterization of overlapping cDNA clones and amino acid sequence analysis.
- Nuclease S1 mapping to identify different mRNA transcripts.
- Antibody generation and Western blot analysis.
Main Results:
- Identified four overlapping cDNA clones encoding a novel low molecular weight human collagen.
- The collagen sequence showed similarities to type IV collagen but had a shorter C-terminal domain (18 amino acids).
- Nuclease S1 mapping revealed two distinct mRNA transcripts differing by 36 nucleotides, encoding four additional Gly-Xaa-Yaa repeats.
- Western blot analysis detected two polypeptides (Mr 67,000 and Mr 62,000) sensitive to bacterial collagenase in tumor cells and fibroblasts.
Conclusions:
- A novel low molecular weight human collagen, distinct from short-chain collagens (IX and X), has been identified.
- The existence of two mRNA variants suggests alternative splicing or transcriptional regulation.
- This collagen is expressed in both tumor cells and normal fibroblasts, indicating a potential role in various cellular processes.