Imre Kovesdi

Cloning, characterization and developmental regulation of two members of a novel human gene family of neurite outgrowth-promoting proteins

This report describes the cloning, expression and characterization of two members of a novel human gene family of proteins, HBNF and MK, which exhibit neurite outgrowth-promoting activity. The HBNF cDNA gene codes for a 168-residue protein which is a precursor for a previously described brain-derived heparin-binding protein of 136 amino acids. The second human gene identified in this study, called MK, codes for a 143-residue protein (including a 22-amino acid signal sequence) which is 46% homologous with HBNF. Complementary DNA constructs coding for the mature HBNF and MK proteins were expressed in bacteria and purified by heparin affinity chromatography. These recombinant proteins exhibited neurite-outgrowth promoting activity, but lacked mitogenic activity. The HBNF gene is expressed in the brain of adult mice and rats, but only minimal expression of MK was observed in this tissue. Different patterns of developmental expression were observed in the embryonic mouse, with MK expression peaking in the brain between days E12 and E14 and diminishing to minimal levels in the adult, while expression of HBNF mRNA was observed to gradually increase during embryogenesis, reaching a maximal level at birth and maintaining this level into adulthood. Expression of these genes was also observed in the human embryonal carcinoma cell line, NT2/D1. Retinoic acid induced the expression of HBNF and MK 6- and 11-fold, respectively, in this cell line. Our studies indicate that HBNF and MK are members of a new family of highly conserved, developmentally regulated genes that may play a role in nervous tissue development and/or maintenance.

HBNF and MK, members of a novel gene family of heparin-binding proteins with potential roles in embryogenesis and brain function

HBNF (heparin-binding neurite-promoting factor) is a heparin-binding protein which is found primarily in the brain and stimulates neurite outgrowth in cultured perinatal neurons. It was also reported to be mitogenic for fibroblasts and endothelial cells but this activity is still controversial. The sequence of HBNF is highly conserved in diverse species suggesting important function. Expression of the HBNF gene in brain tissue appears to be developmentally regulated, increasing during gestation to highest levels around the time of birth. The HBNF gene shows high sequence homology to another gene, MK (midkine). Like HBNF, the MK gene is developmentally regulated, however, high expression occurs in most fetal tissues during mid-gestation. The biological properties of the MK protein are remarkably similar to those of HBNF. The available evidence suggests that HBNF and MK are members of a new family of genes with potential roles in fetal development and in brain function or maintenance.

Heparin-binding neurotrophic factor (HBNF) and MK, members of a new family of homologous, developmentally regulated proteins

A partial rat cDNA clone coding for a novel neurotrophic factor HBNF was isolated. Nucleotide sequence determination, in combination with the known N-terminal sequence of rat HBNF, allowed deduction of the amino acid sequence of the first 102 residues of mature rat HBNF. HBNF shares high structural homology (55%) with the MK protein (Tomomura et al., J. Biol. Chem. 265, 10765, 1990). Complete alignment of 9 cysteine residues suggests further that the two proteins have similar 3-dimensional structures. HBNF was reported to stimulate neurite outgrowth in neurons and to be expressed in a developmentally regulated manner in the rat brain. MK mRNA was found in retinoid acid-induced teratocarcinoma cells and during early development of the mouse embryo, but no biological activity for MK is yet known. These data suggest that HBNF and MK are members of a novel family of structurally and probably functionally related proteins.

Expression of the HBNF (heparin-binding neurite-promoting factor) gene in the brain of fetal, neonatal and adult rat: an in situ hybridization study

HBNF (heparin-binding neurite-promoting factor) and MK (midkine) are members of a newly recognized family of proteins, the expression of which is developmentally regulated. These proteins are expressed highest during fetal development in many tissues but they seem to be rather restricted to the brain in adult animals. Gene expression for these proteins is inducible by retinoic acid in embryonal carcinoma cell lines. They induce neurite outgrowth in cultured neurons, and they are characterized by high sequence conservation between species. While the function(s) of these proteins are unknown, available evidence suggests possible roles in the development and the maintenance of neural tissues. This in situ hybridization study investigates the temporal and spatial expression pattern of the HBNF gene in the brain of developing rats. The HBNF gene is highly expressed in the neuroepithelium and the ependyma from fetal day 15 on. Although most ependymal structures express the gene strongly, a few restricted areas of the ependyma do not express HBNF (ventral part of the fourth ventricle, subcommissural organ). In the brain parenchyma, HBNF is expressed in the thalamo-hippocampal area from fetal day 15 and in the cerebral cortex from fetal day 16, with high expression occurring in the superficial layers of the cortex. The nature of the cells expressing the gene, while difficult to ascertain, is probably glial for the most part. However, certain neurons (in limited areas of the brain parenchyma) and most pial cells (in the meninges), also express the gene. HBNF gene expression decreases sharply a few days after birth. HBNF mRNA is also detectable at fetal days 15 and 16 in the face fetal mesenchyma. In the adult rat brain, the expression of the HBNF gene appears to be restricted to neurons of the hippocampus and of the olfactory bulb and to the superficial layers of the cortex. The structurally related MK gene, though not extensively studied here, shows an entirely different temporal and spatial expression pattern. MK gene is weakly expressed during ontogeny in most brain areas, and in the adult animal, MK mRNA is present only in the choroid plexus. The intense and widely distributed expression of the HBNF gene in several cell populations in the fetus, the progressive spatial and quantitative restriction of HBNF gene expression with brain differentiation, as well as the properties of the protein suggest important and diverse functions for HBNF in cellular interactions and cell differentiation in the developing brain, that must act temporally and spatially by ways distinct from its MK companion molecule.

Isolation from Bovine Brain and Structural Characterization of HBNF, a Heparin-Binding Neurotrophic Factor

A heparin-binding protein with neurotrophic activity for perinatal rat neurons, termed HBNF, was purified to homogeneity from bovine brain utilizing pH 4.5 extraction, ammonium sulfate precipitation, cation exchange and heparin-Sepharose affinity chromatographies, and reverse phase HPLC. In the presence of protease inhibitors during extraction, a protein with an apparent molecular weight of 18 kDa was obtained in a yield of approximately 0.5 mg/kg brain tissue. The amino acid sequence of the first 114 residues of HBNF was determined and found to highly homologous to the cDNA-derived amino acid sequence of human HBNF, a 136-residue protein. Bovine and human HBNFs have identical molecular weights as judged by SDS gel electrophoresis and very similar amino acid compositions. This and overall sequence conservation suggest that bovine HBNF is also a 136 amino acid protein with a calculated molecular weight of approximately 15.5 kDa. The apparent discrepancy between calculated and observed molecular weights of bovine HBNF (and of human HBNF of which the complete sequence is known) is most likely a result of the highly basic nature of HBNF. If protease inhibitors were omitted during tissue extraction, two additional proteins with lower apparent molecular weights and identical N-terminal sequences were isolated, with the smallest forms being the major product. Amino acid analysis showed that the smaller forms correspond to C-terminally truncated HBNFs with calculated molecular weights of 13.6 and 12.4 kDa, lacking approximately 14 and 22 residues. Comparison of the HBNF protein sequence with sequences stored in the Protein Identification Resource/Genbank databases reveals high homology to the translation product of the MK-1 gene, which is retinoic acid-inducible in embryonic carcinoma cells and developmentally expressed during gestation in mice.

Structure/Activity Relationships in Basic FGF

Although the FGFs have been subject to extensive biological studies, only limited progress has been made so far in determining the critical elements of structure-activity relationships in the FGFs. Among the recognized structural elements with potential to affect the biological activity of FGFs are the cysteine residues, and the heparin- and receptor-binding domains. These features have been studied using a variety of experimental approaches, but the available data are inconclusive. For example, ambiguity regarding the presence of a disulfide structure in FGFs was not resolved until the availability of x-ray crystal structure data. Furthermore, the functionally important heparin- and receptor-binding domains have been poorly characterized, with some interpretations being controversial. In this report, we describe a novel fragment of basic FGF (bFGF) with high biological activity [Ser78,96-bFGF(70-153)]. This fragment was generated by pronase treatment of heparin-bound recombinant Glu3,5Ser78,96-bFGF mutant and is active in vitro at an ED50 of about 100 ng/ml. The structure of the fragment and the manner by which it was generated provide additional insight into important aspects of structure-activity relationships in FGFs. Specifically, we conclude that (a) the cysteines in our bFGF mutant do not form a disulfide bond, (b) the high-affinity heparin binding of bFGF critically depends on an intact 3-dimensional structure of the growth factor rather than on specific heparin-binding sequence domains, and (c) the bFGF sequence between residues 70 and 122 is important for high biological activity.