vasoactive-intestinal-peptide and Multiple-Myeloma

Monoclonal human light chains, i.e. Bence Jones proteins, and their recombinant variable fragments (VL) were screened for proteolytic activity using peptide-methylcoumarinamide (peptide-MCA) conjugates and vasoactive intestinal polypeptide (VIP) as substrates. Sixteen of 21 Bence Jones proteins and one of three VL fragments were capable of detectable cleavage of one or more substrates. The magnitude and kinetic characteristics of the activity varied with different substrates. Among the peptide-MCA substrates, the presence of tripeptide or tetrapeptide moieties with a basic residue at the scissile bond generally favored expression of the activity. The influence of N-terminal flanking residue recognition was evident from differing values of Km and kcat (turnover number) observed using different Arg-containing peptide-MCA substrates. Different light chains displayed different kinetic parameters for the same substrate, suggesting unique catalytic sites. Hydrolysis of VIP was characterized by nanomolar Michaelis-Menten constants (Km), suggesting comparatively high affinity recognition of this peptide. The 25-kDa monomer and the 50-kDa dimer forms of one light chain preparation were resolved by gel filtration in 6 M guanidine hydrochloride. Following renaturation, the monomer displayed 51-fold greater peptide-MCA-hydrolyzing activity than the dimer. A renatured VL domain prepared by gel filtration in 6 M guanidine hydrochloride displayed VIP-hydrolyzing activity in the 12.5-kDa peak fractions. These results provide evidence for the proteolytic activity of certain human light chains and imply that this phenomenon may have a pathophysiological significance.

Topics: Amino Acid Sequence; Bence Jones Protein; Catalysis; Coumarins; Endopeptidases; Humans; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Kinetics; Molecular Sequence Data; Multiple Myeloma; Oligopeptides; Peptide Fragments; Substrate Specificity; Vasoactive Intestinal Peptide

Cultured human myeloma cells of the U266 line and leukemic T cells of the Jurkat line bound synthetic [125I]Tyr10-vasoactive intestinal peptide1-28 ([125I]VIP1-28) specifically and with an affinity similar to that of neuroendocrine cells. Specific binding reached equilibrium after 2 h at 22 degrees C for both myeloma cells and T cells, attained a maximum of 57 to 71% of total binding, and was reversed in 1.5 to 3 h by an excess of non-radioactive VIP1-28. Analyses of the ligand concentration-dependence of binding of the ligand concentration-dependence of binding of [125I]VIP1-28 revealed a mean Kd of 7.6 nM for a mean of 41,207 receptors per myeloma cell and 5.2 nM for 12,266 receptors per T cell. The relative affinity of binding of mast cell-derived VIP10-28 free acid and synthetic analogues suggested differences in specificity between lymphocyte and neuroendocrine receptors. Distinct sets of receptors thus appear to mediate the effects of VIP on functions of both antibody-producing cells and T cells.

Topics: Binding, Competitive; Cell Line; Humans; Kinetics; Leukemia, T-Cell; Molecular Structure; Multiple Myeloma; Peptide Fragments; Receptors, Gastrointestinal Hormone; Receptors, Vasoactive Intestinal Peptide; Structure-Activity Relationship; Vasoactive Intestinal Peptide