ABSTRACT
The mouse gene (mHB-EGF) encoding heparin-binding epidermal growth factor-like growth factor was isolated from a mouse 129SVJ genomic library. DNA sequence analysis confirmed that the clone contained six exons (I-VI) and five introns (A-E), and spanned approx. 14 kb of DNA. PCR analysis showed that introns A-E of mHB-EGF are 203 bp, 2.5 kb, 5.5 kb, 825 bp and 272 bp in length, respectively. These results establish that mHB-EGF is similar in organization to human HB-EGF (hHB-EGF). However, DNA sequence analysis of introns A-E of mHB-EGF failed to show significant overall homology with those of hHB-EGF.
Subject(s)
Epidermal Growth Factor/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Genes , Heparin-binding EGF-like Growth Factor , Intercellular Signaling Peptides and Proteins , Introns/genetics , Mice , Molecular Sequence DataABSTRACT
The binding of heparin-binding EGF-like growth factor (HB-EGF) to the epidermal growth factor (EGF) receptor of human endometrial carcinoma cells was compared to that of EGF using an 125I-EGF radioreceptor assay. The inhibitory effect of HB-EGF on 125I-EGF binding was reversed either in the presence of heparin (but not by chondroitin sulfate) or by pre-treating the cells with heparinase. These treatments did not affect the binding of EGF to its receptor. To map potential regions in the HB-EGF molecule that mediate its heparin-dependent interaction with the EGF receptor, HB-EGF peptides were synthesized that were non-homologous to EGF. Accordingly residues 20-25 and 36-41, but not residues 8-19, of HB-EGF were found to be (i) heparin-binding and (ii) modulators of HB-EGF (but not of EGF) binding to the EGF receptor.
Subject(s)
Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Heparin/pharmacology , Peptide Fragments/pharmacology , Polysaccharide-Lyases/pharmacology , Amino Acid Sequence , Binding, Competitive , Chromatography, High Pressure Liquid , Endometrial Neoplasms/metabolism , Female , Heparin Lyase , Heparin-binding EGF-like Growth Factor , Humans , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Radioligand Assay , Tumor Cells, CulturedABSTRACT
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a 22-kDa, O-glycosylated protein. Because recombinant expression systems permitting a detailed analysis of the functional significance of HB-EGF glycosylation have not been described, a recombinant vaccinia virus designed to express HB-EGF was generated by homologous recombination of an intermediate plasmid vector carrying the HB-EGF cDNA and the genome of vaccinia virus and was used to infect HeLa cells. Production of immunoreactive HB-EGF was confirmed by immunofluorescence and radioimmunoprecipitation analysis. Furthermore, the expressed protein was shown to be a secreted, biologically active protein by radioreceptor and DNA synthesis assays of HeLa cell conditioned medium. The recombinant protein was purified from the conditioned medium using heparin-affinity fast protein liquid chromatography followed by C4 reverse-phase high-performance liquid chromatography (RP-HPLC). SDS-PAGE and Western blotting of the RP-HPLC-purified product showed an immunoreactive HB-EGF protein of approximately 22 kDa that was decreased to a 14-kDa protein by treatment with O-glycanase. Amino acid sequencing revealed an N-terminus that was characteristic of native, glycosylated HB-EGF. Interestingly, a Thr residue that is a putative site of O-linked glycosylation failed to be resolved. This system provides a valuable method for evaluating the role of glycosylation in HB-EGF function(s) as well as addressing other questions concerning HB-EGF structure-function relationships.
Subject(s)
Epidermal Growth Factor/metabolism , Vaccinia virus/genetics , Amino Acid Sequence , Chromatography, Affinity , Chromatography, High Pressure Liquid , DNA Primers , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Epidermal Growth Factor/genetics , Fluorescent Antibody Technique , Genetic Vectors/genetics , Glycosylation , HeLa Cells , Heparin/metabolism , Heparin-binding EGF-like Growth Factor , Hexosaminidases/metabolism , Humans , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Molecular Weight , Precipitin Tests , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Analysis , Structure-Activity Relationship , Transfection/geneticsABSTRACT
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially synthesized as a membrane bound protein that is subsequently processed to yield an approximately 74 amino acid secreted product. To investigate the biological activities of HB-EGF and its role(s) in tumor formation, the full-length HB-EGF cDNA was cloned under the regulation of the mouse metallothionein promoter and stably expressed in HB-EGF deficient mouse L cells. HB-EGF immunoreactive proteins of 21 and 24 kDa were observed from transfected MLC lysates, and these lysates exhibited the ability to bind to the EGF receptor, stimulate 3H-thymidine uptake in BALB/c-3T3 cells, and induce anchorage independent growth (AIG) of normal rat kidney (NRK) cells. Furthermore, NRK cells treated with either E. coli-derived or vaccinia virus-derived HB-EGF, as well as NRK cells directly transfected with the HB-EGF construct, demonstrated AIG. We conclude that HB-EGF is a potent growth factor capable of stimulating altered cell growth and anchorage independence.