Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [3 H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [3 H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468 -527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.Tumor cells in vitro and in vivo can develop simultaneous resistance to the lethal effects of a variety of cytotoxic drugs (Endicott and Ling, 1989). This so-called multidrug resistance (MDR) is a major limiting factor for the efficacy of cancer chemotherapy. Currently, a variety of mechanisms are known that can lead to drug resistance, including reduced cellular drug accumulation, increased detoxification, intracellular vesicularization of drugs, altered enzymatic activities, up-or down-regulation of targets, and enhanced DNA repair (Hayes and Wolf, 1990). One important resistance mechanism is the transport out of cancer cells of chemically-unrelated cytotoxic drugs (such as anthracylines, Vinca alkaloids, colchicine, and taxanes) by the integral membrane phosphoglycoprotein P-glycoprotein (P-gp) under ATP hydrolysis, resulting in low and ineffective intracellular drug concentrations (Gottesman and Pastan, 1993). Although considerable progress has been made during the last few years, the mechanism of recognition and transport of such a broad spectrum of compounds is still poorly understood.Reversal of multidrug resistance is of major clinical interest, and MDR-reversing agents called chemosensitizers have been intensively investigated (Raderer and Scheithauer, 1993;Sikic, 1993). The capability of reversi...

Mass spectrometry has been shown in recent years to be a powerful tool to determine accurate molecular masses and sequences of peptides and proteins and post-translational modifications such as glycosylation, phosphorylation, and sulfation. For glycosylation, it has been increasingly recognized to be of pivotal importance to identify whether potential glycosylation sites are actually modified by glycans, because functions of proteins may be modulated or depend on the presence of glycans at specific sites. Several recent reports have established that mass spectrometric techniques such as matrix-assisted laser desorption/ionization or electrospray ionization mass spectrometry (MALDI-TOF or ESI-MS, respectively) with or without preceding HPLC and in combination with PNGase F treatment are suited to analyze whether consensus sequences for N-glycosylation are glycosylated or not. Here we report the mass spectrometric analysis of the six potential N-glycosylation sites of the neural cell adhesion molecule NCAM from adult mouse brain. Unmodified peptides and glycopeptides each carrying a single glycosylation site were generated from NCAM by AspN and trypsin treatment and submitted to reversed-phase HPLC with or without prior enzymatic release of N-glycans. The resulting peptides were analyzed by MALDI-TOF-MS. In addition, high-resolution Fourier transform-ion cyclotron resonance (MALDI-FTICR) mass spectrometry was performed after in-gel deglycosylation and subsequent trypsin digestion. By using these procedures all six consensus sequences were shown to be glycosylated; the observation of an unmodified peptide with the consensus sequence N-1 indicates only partial glycosylation at this site.

Neurolin is a growth-associated cell surface glycoprotein from goldÐsh and zebra Ðsh which has been shown to be involved in axonal path-Ðnding in the goldÐsh retina and suggested to function as a receptor for axon guidance molecules. Being a member of the immunoglobulin superfamily of cell adhesion proteins, neurolin consists of Ðve N-terminal extracellular immunoglobulin (Ig)-like domains, a transmembrane and a short cytoplasmatic domain. Repeated injections of polyclonal Fab fragments against neurolin and of monoclonal antibodies against either Ig domains cause path-Ðnding errors and disturbance of axonal fasciculation. In order to obtain a complete structural characterization and a molecular basis for structure-function determination, recombinant neurolin with the complete extracellular part but lacking the transmembrane and cytoplasmatic domain was expressed in Chinese hamster ovary (CHO) cells (CHO-neurolin). The isolation of CHO-neurolin was carried out by Ni-affinity chromatography and subsequent high-performance liquid chromatography (HPLC). An exact molecular mass determination was obtained by matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and revealed 60.9 kDa, which suggested that ¿10 kDa are due to glycosylation. The predicted molecular mass is 51.5 kDa, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) yielded an apparent molecular mass of 72 kDa. Gel shift assays using SDS-PAGE and Western blot analysis with anti-neurolin antibodies provided consistent molecular mass data. The complete primary structure and N-glycosylation patterns were identiÐed using speciÐc lectin assays, MALDI/MS peptide mapping analysis by proteolytic and in-gel digestion, electrospray ionization MS and MALDI/MS in combination with speciÐc glycosidase degradation. HPLC isolation of glycosylated peptide fragments and MS after selective deglycosylation revealed heterogeneous glycosylations at all Ðve N-glycosylation consensus sites. All attached N-glycans are of the complex type and show a mainly biantennary structure ; they are fucosylated with a(2,3)-terminal neuraminic acid. These data serve as a Ðrst detailed model to characterize the molecular recognition structures exhibited by the extracellular domains.

Axonin-1 is an axon-associated cell adhesion protein which exists as a membrane-attached form via a glycosylphosphatidyl-inositol anchor (GPI) and a secreted form. Axonin-1 promotes and regulates neurite outgrowth and plays a critical role as a growth cone sensor molecule in axonal pathways. The cDNA of axonin-1 from chicken codes for 1036 amino acids, including a hydrophobic Nterminal signal sequence and a C-terminal signal peptide. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SOS-PAGE) of native chicken axonin-1 showed an apparent molecular weight of 130-140 kDa. The characterisation of intact axonin-1 by matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) yielded molecular masses of 122,750 and 125,100 Da which are consistent with the GPI-modified and an unmodified secreted form. The mass difference between the calculated molecular mass based on the amino acid sequence (107,640 Da) suggests modifications ofup to 15 kDa which are assumed to be mainly due to glycosylation. In this study, the detailed structural characterisation of axonin-1 in its secreted GPI-anchorless form was carried out. Mass spectrometric peptide mapping analyses using the endoproteinases Lys-C and Asp-N revealed the complete amino acid sequence, the N-and C-terminal heterogeneities and, in conjunction with reductive cleavage, the intramolecular disulphide-loop pattern. Furthermore, the preliminary results on the location of N-glycosylation sites could be obtained. In addition, proteolytic fragments were isolated by high performance liquid chromatography (HPLC), identified by MALDI-MS and further digested with trypsin. These results revealed the presence of a blocked N-terminus (pyroglutamyl pQ 22 ) and provided the processed C-terminus as M 1004 for the native, GPI-unmodified form of axonin-1.