No abstract

No abstract

Apoptosis-inducing ligand 2 (Apo2L), also called tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers programmed cell death in various types of cancer cells but not in most normal cells. Apo2L/TRAIL is a homotrimeric protein that interacts with five receptors: death receptor 4 (DR4) and DR5 mediate apoptosis activation, whereas decoy receptor 1 (DcR1), DcR2, and osteoprotegerin counteract this function. Many cancer cell lines express both DR4 and DR5, and each of these receptors can initiate apoptosis independently of the other. However, the relative contribution of DR4 and DR5 to ligand-induced apoptosis is unknown. To investigate this question, we generated death receptor-selective Apo2L/TRAIL variants using a novel approach that enables phage display of mutated trimeric proteins. Selective binding to DR4 or DR5 was achieved with three to six-ligand amino acid substitutions. The DR4-selective Apo2L/TRAIL variants examined in this study showed a markedly reduced ability to trigger apoptosis, whereas the DR5-selective variants had minimally decreased or slightly increased apoptosis-inducing activity. These results suggest that DR5 may contribute more than DR4 to Apo2L/TRAIL-induced apoptosis in cancer cells that express both death receptors. Apo2L1 (or TRAIL), a member of the tumor necrosis factor (TNF) superfamily, induces apoptosis in a broad spectrum of human cancer cell lines while sparing most normal cells (1). Apo2L/TRAIL triggers apoptosis through binding to the death receptors DR4 (2) and/or DR5 (3, 4). These receptors contain a cytoplasmic death domain that recruits adaptor molecules involved in caspase activation (5). In addition to these two signaling receptors, Apo2L/TRAIL binds to three decoy receptors that inhibit apoptosis induction: DcR1, DcR2, and OPG. DcR1 and OPG lack a cytoplasmic domain, whereasDcR2 has a truncated death domain that is non-functional for apoptosis initiation (1, 6). Like several other members of the TNF superfamily, Apo2L/TRAIL is synthesized as a type II transmembrane protein that can be proteolytically cleaved to release a soluble, homotrimeric molecule. A recombinant version of soluble homotrimeric Apo2L/TRAIL (residues 114 -281) induces apoptosis in various cancer cell lines but not in normal cells (7,8). Administration of soluble Apo2L/TRAIL in mouse xenograft models of human cancer results in marked anti-tumor activity without systemic toxicity (7-9). These results have prompted further evaluation of Apo2L/TRAIL as a potential therapeutic agent for human cancer.Structural studies show that homotrimeric TNF superfamily ligands bind three receptor molecules (10 -12), suggesting that the basic functional signaling unit is trimeric. This notion is further supported by the trimeric structure of certain signaling adaptor molecules that act downstream of the receptors, such as the TNF receptor-associated factors (13,14). Further crosslinking of receptors beyond the trimeric unit in some cases can lead to stronger signal initiation; this can be modeled by ...

Biotechnological companies and regulatory agencies are pursuing the complete characterization of protein therapeutics in every detail as a means to mitigate risks of product quality related safety issues. During the characterization of a recombinant humanized monoclonal antibody (referred to as rhuMAb), electrospray mass spectrometric analysis suggested that the light chain was highly glycated. The glycated and unglycated materials, separated using boronate affinity chromatography, were fully characterized using tryptic peptide mapping and tandem mass spectrometry. Using an automatic SEQUEST search of the single protein database for this antibody and extensive manual investigations of the mass spectra of the matched peptides, multiple tentative glycation sites in the light and heavy chains were observed in the highly glycated (>53%) samples. A predominant glycation site was identified and confirmed to be lysine 49 on the light chain, by performing extensive sequence analysis on an isolated glycated peptide utilizing Edman degradation analysis and MALDI-TOF/TOF mass spectrometry. Sequence alignments of rhuMAb with 12 other recombinant monoclonal antibodies and computer modeling of the Fab part of rhuMAb suggest that the unusually high level of glycation of lysine residue 49, which is located adjacent to the second complementarity-determining region (CDR2) in the light chain, is due to a spatial proximity effect in catalyzing the Amadori rearrangement by aspartic acid residue 31 in the CDR1 on the light chain.

No abstract