ABSTRACT
The alpha-fetoprotein (AFP) binding protein, a putative AFP receptor, is a tumour marker that is present on the surfaces of malignant cells. AFP enters cells through receptor-mediated endocytosis. The recombinant C-terminal fragment of AFP (AFP-3BC, which consists of amino acid residues 473-596) was obtained by the expression in Escherichia coli. AFP-3BC was shown to be bound specifically to the AFP putative receptor on tumour cells and accumulated by endocytosis in these cells in a similar manner to that of full-length human AFP. In lymphocytes, the binding and endocytosis of AFP-3BC were absent. Thus, the AFP receptor binding site was shown experimentally to be located within the AFP-3BC sequence. A conjugate of synthesised AFP-3BC with the antitumour antibiotic doxorubicin (DOX-AFP-3BC) demonstrated high antitumour activity in vitro. Thus, AFP-3BC can be used successfully as a vector for the targeted selective delivery of drugs into tumour cells.
Subject(s)
Antineoplastic Agents/pharmacology , Doxorubicin/pharmacology , Peptide Fragments/metabolism , Receptors, Peptide/metabolism , alpha-Fetoproteins/metabolism , Amino Acid Sequence , Antibiotics, Antineoplastic/pharmacokinetics , Antibiotics, Antineoplastic/pharmacology , Binding Sites , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Doxorubicin/pharmacokinetics , Drug Delivery Systems/methods , Endocytosis , Escherichia coli/genetics , Escherichia coli/metabolism , Genetic Vectors/genetics , Genetic Vectors/metabolism , Humans , Lymphocytes/drug effects , Lymphocytes/metabolism , MCF-7 Cells , Molecular Sequence Data , Protein Binding/genetics , Receptors, Peptide/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , alpha-Fetoproteins/geneticsABSTRACT
Adiponectin (Adn) is a protein that circulates in the blood in several oligomeric forms, namely low-, medium-, and high-molecular-weight forms. Adn may serve as a risk factor for type 2 diabetes mellitus (T2DM). The aims of this work were (1) to produce monoclonal antibodies (MAbs) specific to different Adn oligomeric forms, (2) to design immunoassays suitable for measuring the Adn forms present in human blood, and (3) to investigate the changes in Adn forms that occur in patients with T2DM. Gel filtration, fluoroimmunoassays, and Western blotting were utilized as major techniques in this study. MAbs recognizing various oligomeric forms of Adn were obtained. Complexes between Adn and complement component C1q and between the low molecular weight form of Adn and albumin were described in human blood. A decrease in the total Adn and Adn-albumin complex levels in the blood of patients with T2DM and no difference in the levels of the Adn-C1q complex in comparison with healthy volunteers were demonstrated. An Adn94-Adn63 fluoroimmunoassay was selected as the technique that most accurately measured the mass ratio of Adn oligomers in blood samples, and an Adn214-Adn27 assay that measured the low-molecular-weight form of Adn only.
Subject(s)
Adiponectin/blood , Diabetes Mellitus, Type 2/blood , Immunoconjugates/blood , Adiponectin/chemistry , Adult , Aged , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/chemistry , Case-Control Studies , Complement C1q/chemistry , Female , Humans , Immunoconjugates/chemistry , Male , Mice , Middle Aged , Molecular Weight , Protein Binding , Protein Isoforms/blood , Protein Isoforms/chemistry , Serum Albumin/chemistryABSTRACT
BACKGROUND: The appearance of B-type natriuretic peptide (BNP) in the blood is ultimately caused by proteolytic processing of its precursor, proBNP. The mechanisms leading to the high plasma concentration of unprocessed proBNP are still poorly understood. The goals of the present study were to examine whether processing of proBNP takes place in the circulation and to evaluate the clearance rate of proBNP and proBNP-derived peptides. METHODS: We studied the processing of human proBNP in the circulation and the clearance rate of proBNP and proBNP-derived peptides (BNP and N-terminal fragment of proBNP, NT-proBNP) in rats by injecting the corresponding peptides and analyzing immunoreactivity at specific time points. Glycosylated and nonglycosylated proBNP and NT-proBNP were used in the experiments. We applied immunoassays, gel filtration, and mass spectrometry (MS) techniques to analyze the circulation-mediated processing of proBNP. RESULTS: ProBNP was effectively processed in the circulation into BNP (1-32) and various truncated BNP forms as confirmed by gel filtration and MS analysis. Glycosylation of proBNP close to the cleavage-site region suppressed its processing in the circulation. The terminal half-life for human glycosylated proBNP was 9.0 (0.5) min compared with 6.4 (0.5) min for BNP. For NT-proBNP, the terminal half-lives were 15.7 (1.4) min and 15.5 (1.3) min for glycosylated and nonglycosylated forms, respectively. CONCLUSIONS: In rats, processing of human proBNP to active BNP occurs in the circulation. The clearance rate of proBNP is quite similar to that of BNP. These observations suggest that peripheral proBNP processing may be an important regulatory step rather than mere degradation.
