We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production.

We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.

Signaling through receptor protein tyrosine phosphatases (RPTPs) can influence diverse processes, including axon development, lymphocyte activation, and cell motility. The molecular regulation of these enzymes, however, is still poorly understood. In particular, it is not known if, or how, the dimerization state of RPTPs is related to the binding of extracellular ligands. Protein tyrosine phosphatase (PTP) is an RPTP with major isoforms that differ in their complements of fibronectin type III domains and in their ligand-binding specificities. In this study, we show that PTP forms homodimers in the cell, interacting at least in part through the transmembrane region. Using this knowledge, we provide the first evidence that PTP ectodomains must be presented as dimers in order to bind heterophilic ligands. We also provide evidence of how alternative use of fibronectin type III domain complements in two major isoforms of PTP can alter the ligand binding specificities of PTP ectodomains. The data suggest that the alternative domains function largely to change the rotational conformations of the amino-terminal ligand binding sites of the ectodomain dimers, thus imparting novel ligand binding properties. These findings have important implications for our understanding of how heterophilic ligands interact with, and potentially regulate, RPTPs.Many cell-signaling events are regulated through reversible tyrosine phosphorylation of proteins. This phosphorylation cycle is controlled by the counterbalanced actions of two enzyme families, protein tyrosine kinases and protein tyrosine phosphatases (PTPs), each of which has cytoplasmic and receptorlike members. While the regulation and actions of many receptor protein tyrosine kinases (RTKs) are well characterized, our related understanding of the receptor-type PTPs (RPTPs) remains far from complete.Twenty-one human RPTPs have been identified, and highly conserved orthologues and homologues exist in vertebrates and invertebrates (3). Several of these RPTPs have particularly well-documented roles in neural development (16,26,40), cell adhesion, and motility (7, 51). RPTPs are type I transmembrane proteins with varied extracellular domain structures, and in many cases, it is still unclear what their ligands are. It is also unclear how, and in most cases if, such ligands directly control RPTP activity. Moreover, although RTKs are obligate dimers during ligand activation and signaling, dimerization has been demonstrated for only a small number of RPTPs, and we have only a few clues as to how this dimerization is linked to signaling control. Furthermore, it is not clear whether ligand binding is influenced by, or influences, the RPTP dimerization state.The RPTPs PTP␣, Sap-1, and CD45 form dimers normally in the cell, and this dimerization can block the catalytic action of these enzymes (23,24,64). PTP␣ and CD45 may be inhibited by steric constraints, possibly through an inhibitory wedge structure (8, 29), although this is still controversial (35). With PTP␣ and Sap-1, there is also e...

The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high-level antibody secretion. We used GS-NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid-exponential growth phase. These were analysed by 2-D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up-regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell-engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.

Vertebrate nervous system development relies on a multitude of guidance cues to stimulate axonal extension and stable synaptic contacts with targets such as muscles. Interpretation of these environmental signals by growth cones involves multiple receptor classes such as cell adhesion molecules (CAMs) Reversible tyrosine phosphorylation, catalyzed by receptor tyrosine kinases and receptor tyrosine phosphatases, plays an essential part in cell signaling during axonal development. Receptor protein tyrosine phosphatase-r has been implicated in the growth, guidance and repair of retinal axons. This phosphatase has also been implicated in motor axon growth and innervation. Insect orthologs of receptor protein tyrosine phosphatase-r are also implicated in the recognition of muscle target cells. A potential extracellular ligand for vertebrate receptor protein tyrosine phosphatase-r has been previously localized in developing skeletal muscle. The identity of this muscle ligand is currently unknown, but it appears to be unrelated to the heparan sulfate ligands of receptor protein tyrosine phosphatase-r. In this study, we have used affinity chromatography and tandem MS to identify nucleolin as a binding partner for receptor protein tyrosine phosphatase-r in skeletal muscle tissue. Nucleolin, both from tissue lysates and in purified form, binds to receptor protein tyrosine phosphatase-r ectodomains. Its expression pattern also overlaps with that of the receptor protein tyrosine phosphatase-r-binding partner previously localized in muscle, and nucleolin can also be found in retinal basement membranes. We demonstrate that a significant amount of muscle-associated nucleolin is present on the cell surface of developing myotubes, and that two nucleolin-binding components, lactoferrin and the HB-19 peptide, can block the interaction of receptor protein tyrosine phosphatase-r ectodomains with muscle and retinal basement membranes in tissue sections. These data suggest that muscle cell surface-associated nucleolin represents at least part of the muscle binding site for axonal receptor protein tyrosine phosphatase-r and that nucleolin may also be a necessary component of basement membrane binding sites of receptor protein tyrosine phosphatase-r.Abbreviations AP, alkaline phosphatase; CAM, cell adhesion molecule; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; HB-19, 5[Kw(CH 2 N)PR]-TASP; HSPG, heparin sulfate proteoglycan; PLAP, placental alkaline phosphatase; PTP, protein tyrosine phosphatase; RAP, receptor affinity probe; RPTP, receptor protein tyrosine phosphatase; RTK, receptor protein tyrosine kinase.