The crystal structure of chicken egg white cystatin has been solved by X‐ray diffraction methods using the multiple isomorphous replacement technique. Its structure has been refined to a crystallographic R value of 0.19 using X‐ray data between 6 and 2.0A. The molecule consists mainly of a straight five‐turn alpha‐helix, a five‐stranded antiparallel beta‐pleated sheet which is twisted and wrapped around the alpha‐helix and an appending segment of partially alpha‐helical geometry. The ‘highly conserved’ region from Gln53I to Gly57I implicated with binding to cysteine proteinases folds into a tight beta‐hairpin loop which on opposite sides is flanked by the amino‐terminal segment and by a second hairpin loop made up of the similarly conserved segment Pro103I ‐ Trp104I. These loops and the amino‐terminal Gly9I ‐ Ala10I form a wedge‐shaped ‘edge’ which is quite complementary to the ‘active site cleft’ of papain. Docking experiments suggest a unique model for the interaction of cystatin and papain: according to it both hairpin loops of cystatin make major binding interactions with the highly conserved residues Gly23, Gln19, Trp177 and Ala136 of papain in the neighbourhood of the reactive site Cys25; the amino‐terminal segment Gly9I ‐ Ala10I of bound cystatin is directed towards the substrate subsite S2, but in an inappropriate conformation and too far away to be attacked by the reactive site Cys25. As a consequence, the mechanism of the interaction between cysteine proteinases and their cystatin‐like inhibitors seems to be fundamentally different from the ‘standard mechanism’ defined for serine proteinases and most of their protein inhibitors.

Background-Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions.Methods-Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay.Results-A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcα1-3(Fucα1-2)Galβ-containing carbohydrates, and . The lectin exerts antiproliferative activity specific to human leukemic T cells. Conclusions-The protein belongs to the ricin B-like lectin superfamily, and has been designated as Clitocybe nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells.General Significance-CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.

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Cystatin, the protein inhibitor of cysteine proteinases from chicken egg white was purified by a new method. The two major forms with pi 6.5 (Peak I) and 5.6 (Peak II) were separated. Molecular masses of both forms are approx. 12 700 Da as determined by gel chromatography; Form A from Peak I has a molecular mass of 12 191 Da as calculated from its aminoacid sequence. The complete amino-acid sequence of Form A was determined by automated solid-phase Edman degradation of the whole inhibitor and its cyanogen bromide fragments. It contains 108 amino-acid residues. Form B from Peak II represents an elongation of Form A by 8 amino-acid residues at the N-terminus. Cystatin contains four cysteine residues, presumably forming two disulphide bridges. Comparison of the amino-acid sequences and near ultraviolet circular dichroism spectra of stefin, the cysteine proteinase inhibitor from human granulocytes, and cystatin shows that the two proteins are entirely different. According to the primary structures, probably neither proteinase inhibitor is involved in a thiol-disulphide exchange mechanism in the interaction with its target enzyme. Cysteinproteinasen-Inhibitoren, III. Aminosäuresequenz von Cystatin aus HühnereiweißZusammenfassung: Aus Hühnereiweiß wurde mit einer neuen Methode der CysteinproteinasenInhibitor Cystatin isoliert. Zwei Hauptformen mit isoelektrischen Punkten von 6.5 (Peak I) und 5.6 (Peak II) konnten getrennt werden. Die durch Gelchromatographie bestimmte apparente Molekularmasse beträgt für beide Formen rund 12 700 Da; aus der Aminosäuresequenz wurde für die Form A aus Peak I eine Molekularmasse von 12 191 Da berechnet. Durch automatischen Festphasen-Edman-Abbau des ganzen Inhibitors und seiner Bromcyanfragmente wurde die vollständige Aminosäuresequenz der Form A ermittelt (108 Aminosäurereste). Form B aus Peak II ist gegenüber der Form A um 8 Aminosäurereste am NTerminus verlängert. Cystatin enthält 4 Cysteinreste, die wahrscheinlich über Disulfidbrücken verknüpft sind.

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