Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.
Mass spectrometry is now firmly established as a powerful technique for the identification and characterization of proteins when used in conjunction with sequence databases. Various approaches involving stable-isotope labeling have been developed for quantitative comparisons between paired samples in proteomic expression analysis by mass spectrometry. However, interpretation of such mass spectra is far from being fully automated, mainly due to the difficulty of analyzing complex patterns resulting from the overlap of multiple peaks arising from the assortment of natural isotopes. In order to facilitate the interpretation of a complex mass spectrum of such a mixture, such as an MS spectrum of a stable-isotope-enriched ion species, we report on the development of a software application, 'Matching' (web accessible), that enables the automatic matching of theoretical isotope envelopes to multiple ion peaks in a raw spectrum. It is particularly useful for resolving the relative abundances of narrow-split paired peaks caused by enrichment with a stable isotope, such as 18O, 13C, 2H, or 15N.
The web application Isotopica has been developed as an aid to the interpretation of ions that contain naturally occurring isotopes in a mass spectrum. It allows the calculation of mass values and isotopic distributions based on molecular formulas, peptides/proteins, DNA/RNA, carbohydrate sequences or combinations thereof. In addition, Isotopica takes modifications of the input molecule into consideration using a simple and flexible language as a straightforward extension of the molecular formula syntax. This function is especially useful for biomolecules, which are often subjected to additional modifications other than normal constituents, such as the frequently occurring post-translational modification in proteins. The isotopic distribution of any molecule thus defined can be calculated by considering full widths at half maximum or mass resolution. The combined envelope of several overlapping isotopic distributions of a mixture of molecules can be determined after specifying each molecule's relative abundance. The results can be displayed graphically on a local PC using the Isotopica viewer, a standalone application that is downloadable from the sites below, as a complement to the client browser. The m/z and intensity values can also be obtained in the form of a plain ASCII text file. The software has proved to be useful for peptide mass fingerprinting and validating an observed isotopic ion distribution with reference to the theoretical one, even from a multi-component sample. The web server can be accessed at http://bioinformatica.cigb.edu.cu/isotopica and http://coco.protein.osaka-u.ac.jp/isotopica [correction].
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