The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 Å resolution were collected at the ESRF synchrotron-radiation source.
AbstractSecreted as well as membrane-associated eukaryotic proteins are most commonly glycosylated. Saccharides are attached to proteins mainly through N- and O-glycosydic bonds or as part of the glycosylphosphatidyinositolmembrane anchor. In contrast to N-glycosylation, which involves the co-translational transfer in the endoplasmic reticulum (ER) of the glycan portion of Glc3Man9GlcNAc2-PP-dolichol to suitable Asn residues on nascent polypeptides, O-glycosylation begins with the addition of a single monosaccharide. Contrary to N-glycosylation, which involves an asparagine residue in the sequon Asn-Xaa-Thr/Ser (Xaa can be any amino acid except Pro, and it is rarely Cys), no particular sequence motif has been described for O-glycosylation. This may reflect the fact that: (1) the specificity of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase is presently unknown; and (2) seems to be modulated by sequence context, secondary structure, and surface accessibility (1). An internet server, accessible at http://www.cbs.dtu.dk/netOglyc/cbsnetOglyc.html, produces neural network predictions of mucin-type GalNAc O-glycosylation sites in mammalian proteins based on 299 known and verified mucin-type O-glycosylation sites. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Nonclustered sites had a sequence context different from that of clustered sites, and charged residues were disfavored at position −1 and +3.KeywordsNeutral SugarSequence ContextMALDI Mass SpectrometryFPLC SystemAutomate Amino Acid AnalyzerThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.