Angelo G. Gambarini

Mitogenic activity of acidic fibroblast growth factor is enhanced by highly sulfated oligosaccharides derived from heparin and heparan sulfate

The mitogenic activity of acidic fibroblast growth factor (aFGF) is potentiated by the highly sulfated hexasaccharide [IdoUA,2S-GlcNS,6S]2-[GlcUA-GlcNS,6S] the structural repetitive unit of lung heparin chains. On a mass basis, the effect of both heparin and oligosaccharide are equivalent whereas on a molar basis, heparin, which contains about seven hexasaccharide repeats, is more efficient. On the other hand, a pentasulfated tetrasaccharide or di- and trisulfated disaccharides are much less effective in potentiating aFGF activity than the hexasaccharide. If the growth factor is pre-incubated with the hexasaccharide at pH 7.2 and then exposed to pH 3.5 the 306/345 nm fluoresence ratio is similar to that of native aFGF indicating that the oligosaccharide stabilizes a native conformation of the protein. Heparan sulfates extracted from various mammalian tissues were also able to potentiate aFGF mitogenic activity. On a mass basis they were in general less efficient than heparin; however, heparan sulfate prepared from medium conditioned by 3T3 fibroblasts is more efficient than heparin both on a mass and molar basis. A highly sulfated oligosaccharide isolated after digestion of pancreas heparan sulfate with heparitinase I is more active than the intact molecule, reaching a potentiating effect equivalent to that of lung heparin, whereas an N-acetylated oligosaccharide isolated after nitrous acid degradation is inactive. These data suggest that the mitogenic activity of aFGF is primarily potentiated by interacting with highly sulfated regions of heparan sulfates chains.

Neuropeptide-Metabolizing Peptidases in Neuro-2a Neuroblastoma and C6 Glioma Cells

Abstract: Mouse Neuro‐2a neuroblastoma and rat C6 glioma cloned cells were screened for neuropeptide‐metabolizing peptidases using a kininase bioassay combined with a time‐course bradykinin‐product analysis, and a fluorimetnc assay for prolyl endopeptidase. The complementary peptide products Arg1→ Phe5/Ser6→ Arg9 and Arg1→ Pro7/Phe8‐Arg9 were released during bradykinin (Arg1‐Pro2‐Pro3‐Gly4‐Phe5‐Ser6‐Pro7‐Phe8‐Arg9) inactivation by homogenates of Neuro‐2a and C6 cells. The 1:1 stoichiometry of the complementary fragments and their high yields, at 10% bradykinin inactivation, demonstrated the sites of hydrolysis. The initial rate of Phe5‐Ser6 bond cleavage was six‐fold higher than that of the Pro7‐Phe8 bond. These sites of cleavage can be attributed to enzymes similar to endqpeptidase A (Phe5‐Ser6) and prolyl endopeptidase (Pro7‐Phe8) on the basis of the specificity and sensitivity to inhibitors of the kininase activity in Neuro‐2a and C6 cell homogenates. Kininase and prolyl endopeptidase specific activities (fmol/min/cell) were 10.5 and 12.4 for Neuro‐2a, and 1.5 and 2 for C6 homogenate, respectively. The recovery of kininase activity was 2.2‐fold higher in the particulate than in the soluble (105,000 g for 1 h) neuronal fraction, whereas the amount of prolyl endopeptidase activity was about the same in both fractions. Kininase and prolyl endopeptidase activities in C6 cells were recovered mostly in the soluble fraction. Prolyl endopeptidase specific activity decreased 10‐fold in serum‐starved Neuro‐2a cultured cells, with no change in activity in similarly treated C6 cells. In contrast, kininase specific activity in both cell types was essentially unaffected on serum‐deprivationinduced differentiation.

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.

Mitogenic activity of peptides related to the sequence of human Fibroblast Growth Factor‐1

Linear synthetic peptides related to the human Fibroblast Growth Factor‐1 (hFGF‐1) segment [112‐147] were tested for their capacity of mimicking FGF mitogenic activity, binding to heparin‐Sepharose columns, stimulating DNA synthesis and competing with hFGF‐1 for the cellular receptors. The results obtained indicated that the activity of these compounds is dependent on the presence of the sequence WFVGLK in their structures. The affinity for the cellular receptors increased when this sequence was elongated in order to incorporate amino acid residues that are important for FGF‐heparin binding.