Isolation and structural and functional characterization of two stable peptic fragments of human alpha-fetoprotein.
Short-time limited peptic hydrolysis of ligand-free human alpha-fetoprotein (AFP) gave two main fragments with molecular masses of 38 and 32 kDa, which had been produced by splitting of the molecule at the position Leu(312)-Asn(313). A more prolonged proteolysis led to the further degradation of these fragments and appearance of highly proteolytically resistant 23-kDa (P23) and 26-kDa (P26) fragments, corresponding to N- and C-terminal parts of the AFP molecule, respectively. Comparative study of intact free of ligands AFP and isolated stable P23 and P26 fragments by circular dichroism, differential scanning calorimetry, and immunoprecipitation techniques demonstrated that these fragments conserved native secondary, tertiary; and antigenic structure, characteristic of the intact molecule. It was concluded that, free of ligands, the AFP molecule could be considered as a three-domain molecule, in which two compact rigid domains (N-terminal domain I and C-terminal domain III) are connected by relatively labile domain II. The structure of domain II could be approximated by a "molten globule" state, characterized by the absence of rigid tertiary structure but having a pronounced secondary structure. Tumor-suppressive activity via induction of apoptosis was recently shown for AFP [Dudich, E. I., et al. (1998) Tumor Biol. 19, 261-272]. We studied here the ability of isolated proteolytic AFP fragments to induce apoptosis in the AFP-sensitive Raji cell line, to determine possible localization of the active site responsible for apoptosis signaling. Unlike intact AFP, neither isolated fragments nor their equimolar mixture was able to induce apoptosis in a human lymphoma Raji cell line. However, it was demonstrated that both fragments P23 or P26 and their equimolar mixture P23 + P26 operated synergistically with intact AFP in suppression of Raji cell proliferation. These data suggested that two structurally determined requirements are necessary for AFP-mediated triggering of apoptosis: (i) dimerization of AFP to form the heterodimeric complex of C- and N-terminal domains and (ii) participation of the central part of AFP molecule (domain II).
Dudich
I
I
Institute of Engineering Immunology, Lyubuchany, Moscow Region, 142380, Russia.
Tokhtamysheva
N
N
Semenkova
L
L
Dudich
E
E
Hellman
J
J
Korpela
T
T
D000941:Antigens / Q000737:chemistry / Q000302:isolation & purification; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005312:Fetal Blood / Q000737:chemistry; D006131:Growth Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D006868:Hydrolysis; D005779:Immunodiffusion; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D010449:Peptide Mapping; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D000509:alpha-Fetoproteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*
D000941:Antigens; D000970:Antineoplastic Agents; D006131:Growth Inhibitors; D010446:Peptide Fragments; D000509:alpha-Fetoproteins; D010434:Pepsin A