br Data A summary of the main

Data A summary of the main information regarding number of search inputs, PSMs, peptide IDs, and protein IDs is reported in Table 1. As it is possible to observe, for one of the samples only two SCX fractions were obtained. For this sample the amount of apoplastic proteins that were retrieved from the procedure described above (Section 2.2) was too low to allow a deeper fractionation. The number of protein and peptide IDs from this biological replicate reflect the fact that a lower amount of material was analyzed. Nevertheless, LC–MS/MS analyses led to the identification of a total of 329 proteins; of them, 208 were found in at least two biological replicates. We restricted our analysis to the 118 proteins that were either apoplastic or unlocalized (based on the Gene Ontology assignment for cellular compartmentalization; 〈www.uniprot.org〉 [8], accounting for approximately 57% of the total. The choice of including unlocalised proteins may represent a potential risk of considering as apoplastic some proteins that are not; however, we decided to be less conservative since several evidences in literature point to the occurrence of unconventional secreted proteins that are not predicted as such by bioinformatics tools [9,10]. The variations considered were further restricted to proteins exhibiting an at least ±50% fold change in expression. Differentially expressed proteins are listed in Tables 2–5 and compared in the Venn diagram shown in Fig. 3. This diagram shows that a subset of proteins are altered both as a consequence of the ggt1 mutation, and of the UV-B treatment. These proteins are involved in ROS metabolism (as superoxide dismutase At4g25100) and in very remodeling; one is a Leucine-rich repeat-containing protein (At1g33590), which is associated to the plasma membrane and is likely to act as a receptor. This comparative analysis lead to the hypothesis that the gamma-glutamyl cycle may participate in ROS-mediated environmental stress sensing, by transferring redox signals arising in the apoplast to the inner compartments [1,4,11].
Acknowledgements The authors wish to thank the “Cassa di Risparmio di Padova e Rovigo” (CARIPARO) Foundation for funding the acquisition of the LTQ-Orbitrap XL mass spectrometer. Funding by MURST ex-60% (AM), University of Padova, and from CIB (Consorzio interuniversitario per Biotecnologie) is also gratefully acknowledged.
Specifications Table Value of the data
1. Data, experimental design, materials and methods Multiple Myeloma (MM) is an incurable malignancy of antibody-secreting B cells (plasma cells) with a mean life expectancy of 5 years from diagnosis [1]. We generated a chimeric mouse/human antibody (J22.9-xi) against CD269 (BCMA), a plasma membrane receptor expressed nearly exclusively in plasma cells; the antibody shows potent cell killing activity on MM cells in vitro and anti-tumor activity in vivo. Surface Plasmon Resonance Spectrometry (SPR) measurements gave an exceptionally high affinity of J22.9-xi for BCMA of 50 pM [2]. Although higher affinity antibody:antigen interactions have been achieved by protein engineering/selection techniques [3], the J22.9-xi interaction exceeds the low nanomolar affinity range typical of therapeutic antibodies [4,5] (see also, for example, Herceptin at 5nM [6] and Rituximab at 8nM [7]) by approximately 100-fold. A protruding loop in the antigen BCMA (the DxL loop) with Leu17 at its apex is critical for binding to the native BCMA ligands, B cell Activating Factor of the TNF Family (BAFF) and A PRoliferation Inducing Ligand (APRIL) [8], both of which have a hydrophobic cavity that packs tightly around Leu17. Our 1.89Å X-ray structure of a J22.9-xi Fab fragment in complex with BCMA shows that J22.9-xi also targets the DxL loop (Fig. 1A). However, the Leu17 binding cavity in J22.9-xi is substantially larger than the Leu17 side chain and provides only limited hydrophobic interactions between Leu17 and the VL of the antibody, primarily with Tyr91; the remainder of the binding cavity is filled with 6 water molecules that pack against the side of Leu17 opposite the Tyr91 interaction (Fig. 1A, B and C). This high affinity interaction comprises few minor direct contacts between the molecules (4 direct hydrogen bonds and 21 mostly single atom van der Waals contacts) but at least 50 hydrogen bonds involving waters “bridging” interactions between main chain and side chain atoms; 17 of these water molecules are directly in the binding interface (Table 1). Thus, the structure shows how high affinity binding can be achieved with only a small number of direct interactions between the binding partners.