Molecular cloning of human fibroblast hyaluronic acid-binding protein confirms its identity with P-32, a protein co-purified with splicing factor SF2. Hyaluronic acid-binding protein as P-32 protein, co-purified with splicing factor SF2.
The purification of a 68-kDa hyaluronic acid-binding protein (HA-binding protein), a homodimer of 34 kDa that binds specifically to hyaluronic acid, has been reported earlier by us (Gupta, S., Batchu, R.B., and Datta, K. (1991) Eur. J. Cell Biol. 56, 58-67). Here, we report the isolation of a partial cDNA clone from a lambda gt11 cDNA expression library of human skin fibroblast by immuno-screening with HA-binding protein antiserum. The internal polypeptide sequence (83 residues) of the purified hyaluronic acid-binding protein is identical to the predicted protein sequence derived from hyaluronic acid-binding protein cDNA, suggesting the authenticity of the clone. Interestingly, this hyaluronic acid-binding protein cDNA sequence has complete homology with the cDNA sequence of a protein P-32, co-purified with the human pre-mRNA splicing factor SF2 (Krainer, A.R., Mayeda, A., Kozak, D., and Binns, G. (1991) Cell 66, 383-394). Furthermore, the data on the N-terminal sequence of hyaluronic acid-binding protein and the predicted polypeptide of P-32 revealed the identical coding sequence of 209 amino acids for both the proteins. As the identity and functional characterization of P-32 have not yet been reported, P-32 cDNA was expressed in Escherichia coli, and the recombinant P-32 protein was purified by hyaluronic acid-Sepharose affinity chromatography. The recombinant P-32 protein showed immunocross-reactivity with the polyclonal antibodies raised against HA-binding protein. The predicted amino acid sequence of the protein fulfilled the minimal criteria for binding to hyaluronic acid, i.e. two basic amino acids flanking a seven-amino acid stretch, as reported for other hyaluronic acid affinity of the recombinant P-32 protein was confirmed by biotinylated hyaluronic acid binding assay. The binding of recombinant P-32 protein to biotinylated hyaluronic acid binding assay. The binding of recombinant P-32 protein to biotinylated hyaluronic acid can be competed only with excess unlabeled hyaluronic acid, confirming its specificity toward hyaluronic acid. All these results suggest that both P-32, co-purified with the human pre-mRNA splicing factor SF2, and 34-kDa hyaluronic acid-binding protein reported by us are the same protein and that it is a new member of the hyaluronic acid-binding protein family, the "hyaladherins."
Deb
T B
TB
Biochemistry Laboratory, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India.
Datta
K
K
D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000737:chemistry; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D006820:Hyaluronic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D016601:RNA-Binding Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000068103:Serine-Arginine Splicing Factors
D015704:CD4 Antigens; D017931:DNA Primers; D018076:DNA, Complementary; D018960:Hyaluronan Receptors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D006820:Hyaluronic Acid