Secretion and biological activities of heparin-binding growth-associated molecule. Neurite outgrowth-promoting and mitogenic actions of the recombinant and tissue-derived protein.
The cDNA for the developmentally regulated, neurite outgrowth-promoting protein HB-GAM (heparin-binding growth-associated molecule) was recently cloned and shown to encode a novel lysine-rich sequence that is homologous with retinoic acid-induced sequences suggested to function in cell differentiation (Merenmies, J., and Rauvala, H. (1990) J. Biol. Chem. 265, 16721-16724). The same sequence was found for the mitogenic and neurite outgrowth-promoting protein pleiotrophin (Li, Y.-S., Milner, P. G., Chauhan, A. K., Watson, M. A., Hoffman, R. M., Kodner, C. M., Milbrandt, J., and Deuel, T. F. (1990) Science 250, 1690-1694). In this study, we have constructed a recombinant baculovirus using the cDNA that encodes the putative preprotein of HB-GAM. The putative secretion signal of HB-GAM is cleaved off in the baculovirus expression system, and the recombinant protein is rapidly secreted to the culture medium. Recombinant HB-GAM purified from the culture medium retains the biochemical characteristics and the neurite outgrowth-promoting activity found for the tissue-derived protein. Studies on the neurite outgrowth-promoting activity suggest that HB-GAM functions as an extracellular matrix-associated protein that enhances axonal growth in perinatal cerebral neurons of the rat. Since the same predicted amino acid sequence has been ascribed to a mitogenic protein, mitogenic activities of the recombinant HB-GAM and of tissue-derived HB-GAM fractions were also studied. Recombinant HB-GAM did not display any significant mitogenic activity, suggesting that tissue-derived HB-GAM preparations may contain other heparin-binding mitogenic factors. We identified in brain-derived HB-GAM fractions a 17-kDa protein (p17) that is detached from heparin by a slightly higher salt concentration as compared to HB-GAM. We suggest that p17 is structurally distinct from HB-GAM and responsible for the mitogenic actions of tissue-derived HB-GAM fractions.
Raulo
E
E
Institute of Biotechnology, University of Helsinki, Finland.
Julkunen
I
I
Merenmies
J
J
Pihlaskari
R
R
Rauvala
H
H
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism* / Q000502:physiology; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006133:Growth Substances / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007313:Insecta; D051379:Mice; D008807:Mice, Inbred BALB C; D008934:Mitogens; D008969:Molecular Sequence Data; D016501:Neurites / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D016207:Cytokines; D006133:Growth Substances; D008934:Mitogens; D011994:Recombinant Proteins; C066845:pleiotrophin; D004247:DNA