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Cloning, expression and characterisation of a new human low Mr phosphotyrosine protein phosphatase originating by
A Modesti1, R Marzocchini, G Raugei
1Department of Biochemical Sciences, University of Florence, Italy.
Abstract:
RT-PCR experiments on RNA from K562 and HepG2 cells and from human placenta led to the isolation of a novel cDNA, a further alternative splicing product of the primary transcript of low Mr phosphotyrosine phosphatase (LMW-PTP), already known to produce isoforms 1 and 2. This new transcript represents 15-20% of the total LMW-PTP mRNA in the cell. This novel cDNA codifies for a protein that we have named SV3 (splicing variant 3): the deduced protein sequence presents the first 49 residues identical to those of isoform 1, followed by 24 unrelated amino acids, due to a frameshift introduced at the novel exon-exon boundary. The SV3 protein, expressed in E. coli is enzymatically inactive, most probably because unfolded, as suggested by far-UV circular dichroism (CD) experiments. SV3 protein appears to possess the characteristics of an unstructured polypeptide chain lacking the packing of side chain residues and the secondary structure level that are typical of globular proteins. This protein could represent an inactive variant of the human LMW-PTP.
Insights
Researchers discovered a new splicing variant (SV3) of the low molecular weight phosphotyrosine phosphatase (LMW-PTP) in human cells. This variant codes for an enzymatically inactive protein, likely due to its unstructured nature.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Low molecular weight phosphotyrosine phosphatase (LMW-PTP) is known to produce multiple isoforms.
- Alternative splicing is a key mechanism for generating protein diversity from a single gene.
Purpose of the Study:
- To identify and characterize novel alternative splicing products of the LMW-PTP gene.
- To investigate the structural and functional properties of a newly identified LMW-PTP variant.
Main Methods:
- RT-PCR was used to isolate novel cDNA from K562 cells, HepG2 cells, and human placenta.
- Protein expression in E. coli and subsequent characterization using far-UV circular dichroism (CD) were performed.
Main Results:
- A novel cDNA, representing 15-20% of total LMW-PTP mRNA, was isolated.
- This cDNA encodes a protein named splicing variant 3 (SV3), with a unique sequence due to a frameshift.
- Expressed SV3 protein was found to be enzymatically inactive and exhibited characteristics of an unstructured polypeptide chain.
Conclusions:
- SV3 is a novel, alternatively spliced variant of human LMW-PTP.
- The SV3 protein is likely an inactive variant due to its unfolded and unstructured nature.
- This finding expands the understanding of LMW-PTP isoforms and their functional implications.