Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody.

A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.

Two forms of mouse syntrophin, a 58 kd dystrophin-associated protein, differ in primary structure and tissue distribution.

Syntrophin, a 58 kd extrinsic membrane protein, is concentrated at postsynaptic sites at the neuromuscular junction and may be involved in clustering acetylcholine receptors. In muscle and nonmuscle tissues, syntrophin is associated with dystrophin, utrophin, and two homologs of the dystrophin carboxy-terminal region. We have isolated three cDNAs encoding Torpedo and mouse syntrophins. The Torpedo cDNA encodes a full-length protein, and on Northern blots recognizes a 3.5 kb mRNA. The two mouse syntrophin cDNAs are products of separate genes but encode proteins that share 50% identity. Syntrophin-1 mRNA (2.2 kb) is expressed at highest levels in skeletal muscle. Syntrophin-2 mRNAs (2.2, 5.0, and 10 kb) are expressed in all mouse tissues examined. These patterns of expression suggest that syntrophin-1 and syntrophin-2 may associate with different members of the dystrophin family.

Association of the Mr 58,000 postsynaptic protein of electric tissue with Torpedo dystrophin and the Mr 87,000 postsynaptic protein.

Dystrophin was purified by immunoaffinity chromatography from detergent-solubilized Torpedo electric organ postsynaptic membranes using monoclonal antibodies. A major doublet of proteins at Mr 58,000 and minor proteins at Mr 87,000, Mr 45,000, and Mr 30,000 reproducibly copurified with dystrophin. The Mr 58,000 and Mr 87,000 proteins were identical to previously described peripheral membrane proteins (Mr 58,000 protein and 87,000 protein) whose muscle homologs are associated with the sarcolemma (Froehner, S. C., Murnane, A. A., Tobler, M., Peng, H. B., and Sealock, R. (1987) J. Cell Biol. 104, 1633-1646; Carr, C., Fischbach, G. D., and Cohen, J. B. (1989) J. Cell Biol. 109, 1753-1764). The copurification of dystrophin and Mr 58,000 protein was shown to be specific, since dystrophin was also captured with a monoclonal antibody against the Mr 58,000 protein but not by several control antibodies. The Mr 87,000 protein was a major component (along with the Mr 58,000 protein) in material purified on anti-58,000 columns, suggesting that the Mr 58,000 protein forms a distinct complex with the Mr 87,000 protein, as well as with dystrophin. Immunofluorescence staining of skeletal and cardiac muscle from the dystrophin-minus mdx mouse with the anti-58,000 antibody was confined to the sarcolemma as in normal muscle but was much reduced in intensity, even though immunoblotting demonstrated that the contents of Mr 58,000 protein in normal and mdx muscle were comparable. Thus, the Mr 58,000 protein appears to associate inefficiently with the sarcolemmal membrane in the absence of dystrophin. This deficiency may contribute to the membrane abnormalities that lead to muscle necrosis in dystrophic muscle.

Localization of dystrophin relative to acetylcholine receptor domains in electric tissue and adult and cultured skeletal muscle.

Two high-affinity mAbs were prepared against Torpedo dystrophin, an electric organ protein that is closely similar to human dystrophin, the gene product of the Duchenne muscular dystrophy locus. The antibodies were used to localize dystrophin relative to acetylcholine receptors (AChR) in electric organ and in skeletal muscle, and to show identity between Torpedo dystrophin and the previously described 270/300-kD Torpedo postsynaptic protein. Dystrophin was found in both AChR-rich and AChR-poor regions of the innervated face of the electroplaque. Immunogold experiments showed that AChR and dystrophin were closely intermingled in the AChR domains. In contrast, dystrophin appeared to be absent from many or all AChR-rich domains of the rat neuromuscular junction and of AChR clusters in cultured muscle (Xenopus laevis). It was present, however, in the immediately surrounding membrane (deep regions of the junctional folds, membrane domains interdigitating with and surrounding AChR domains within clusters). These results suggest that dystrophin may have a role in organization of AChR in electric tissue. Dystrophin is not, however, an obligatory component of AChR domains in muscle and, at the neuromuscular junction, its roles may be more related to organization of the junctional folds.

Immunochemical identification of desmin in Torpedo postsynaptic membranes and at the rat neuromuscular junction.

Preparations of acetylcholine receptor-rich (AChR-rich) postsynaptic membranes from electric tissue of electric rays often contain an Mr 55,000 protein (55kD protein) that has not been previously characterized. Using a monoclonal antibody (MAb 1403) against the 55kD protein from Torpedo californica and a pan-specific, anti-intermediate filament antibody (Pruss et al., 1981; Cell 27:419-428), we show that the 55kD protein has the properties expected of Torpedo desmin. By the electron microscope immunogold method applied to perfusion-fixed electric tissue, MAb 1403 labeled only cytoplasmic filaments in the electroplax. These filaments were neither more concentrated nor arranged detectably differently in postsynaptic regions relative to nonpostsynaptic regions. The 55kD protein could also be fractionated away from isolated postsynaptic membranes by gradient centrifugation. The protein is thus a minor component of the postsynaptic membrane in situ and after isolation. On semithin cryosections of rat skeletal muscle, on the other hand, MAb 1403, which recognizes rat desmin but not rat vimentin, gave strong fluorescent labeling of the postsynaptic region, weaker labeling of the Z-line, and still weaker labeling of the cell surface immediately surrounding extra-junctional nuclei. The pattern of postsynaptic labeling suggests that desmin, presumably in the form of intermediate filaments, occurs near the AChR-rich crests of the junctional folds, but is particularly concentrated among and around the ends of the folds. Similar results were obtained with a second monoclonal antibody raised against authentic desmin. These results suggest that desmin intermediate filaments may have an important role in organization of the postsynaptic cytoplasm in rat muscle.

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle.

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.