Mitogenic and in vitro angiogenic activity of human recombinant heparin affin regulatory peptide.
We have previously described the purification of a heparin binding growth factor from adult bovine brain named heparin affin regulatory peptide (HARP), which was identical to an uterus derived growth factor named pleiotrophin and to a developmentally regulated neurite promoting factor named heparin-binding growth associated molecule. However, for yet unclear reasons, the mitogenic activity of this purified polypeptide following isolation from animal tissue extracts is a subject of controversy, due to conflicting and irreproducible data when produced by recombinant DNA technologies in E. coli or insect cells. The purified protein was inactive in mitogenic assays but the natural molecule was active in assay of neurite outgrowth. In order to clarify these conflicting results and to obtain a recombinant protein free from other contaminating heparin-binding growth factors, we have cloned human cDNA encoding human HARP, engineered its expression in NIH 3T3 cells and characterised the resulting recombinant polypeptide. Purified recombinant HARP displayed mitogenic activity for capillary endothelial cells with half-maximal stimulation at approximately 1 ng/ml (55 pM) and induced angiogenesis in an in vitro model. Interestingly, while the NH2 terminal sequence of tissue purified HARP was NH2-GKKEKPEKK, the NH2 terminal sequence of the biologically active recombinant protein was NH2-AEAGKKEKPEKK, corresponding to a three amino acid extended form.
Laaroubi
K
K
Laboratorie de Recherche sur la Croissance Cellulaire, Université Paris Val de Marne, Créteil, France.
Delbé
J
J
Vacherot
F
F
Desgranges
P
P
Tardieu
M
M
Jaye
M
M
Barritault
D
D
Courty
J
J
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D003001:Cloning, Molecular; D017077:Culture Media, Conditioned; D016207:Cytokines / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D051379:Mice; D008934:Mitogens / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009389:Neovascularization, Pathologic; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D014162:Transfection
D002352:Carrier Proteins; D017077:Culture Media, Conditioned; D016207:Cytokines; D018076:DNA, Complementary; D008934:Mitogens; D011994:Recombinant Proteins; C066845:pleiotrophin