Yakov Gluzman

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.

INHIBITION OF HUMAN CYTOMEGALOVIRUS UL80 PROTEASE BY SPECIFIC INTRAMOLECULAR DISULFIDE BOND FORMATION

A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.

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.

Preliminary X-ray crystallographic analysis of a modified basic fibroblast growth factor

Human basic fibroblast growth factor (hbFGF) has been modified, with Ala3 and Ser5 substituted by glutamic acid, and the purified recombinant protein has been crystallized. The crystals are triclinic (space group P1) with unit cell parameters a = 31.0 A, b = 33.6 A, c = 34.7 A, alpha = 88 degrees, beta = 85 degrees, gamma = 76 degrees, and they diffract to at least 2 A.