Dual Affinity Labeling of the Active Site of Human Lysozyme with an N-Acetyllactosamine Derivative: First Ligand Assisted Recognition of the Second Ligand^,

Abstract

Among the three kinds of the 2',3'-epoxypropyl beta-glycoside of disaccharides (GlcNAc-beta1,4-GlcNAc, Gal-beta1,4-GlcNAc, and Man-beta1,4-GlcNAc), the derivative of N-acetyllactosamine (Gal-beta1,4-GlcNAc-Epo) caused the dual labeling of human lysozyme (HL) most efficiently. The labeled HL was crystallized and analyzed by X-ray diffraction methodology. The X-ray analysis located the two Gal-beta1,4-GlcNAc-Epo moieties inside the catalytic cleft of HL. The attachment sites were the side-chain carboxylate groups of the catalytic residues Glu35 and Asp53 in HL. The first Gal-beta1, 4-GlcNAc-Epo moiety occupied virtually the same position as observed in the HL labeled with single Gal-beta1,4-GlcNAc-Epo molecule. The second Gal-beta1,4-GlcNAc-Epo moiety was recognized via the carbohydrate-carbohydrate interaction with the first Gal-beta1, 4-GlcNAc-Epo moiety in addition to the protein-carbohydrate interaction with the "right-side" catalytic cleft of HL through a number of hydrogen bonds including water-mediated ones as well as many van der Waals contacts. The two N-acetylglucosamine residues stacked with each other, while the two rings of galactose residues approximately shared the same plane. The dual labeling with two Gal-beta1,4-GlcNAc-Epo molecules was supposed to have occurred sequentially, which was accompanied with the alteration to the pKa of Glu35 derived from the esterification of Asp53 in the first labeling. Both asymmetric carbons in the connection parts between HL and N-acetyllactosamine moieties showed the same stereoconfiguration derived from the reaction with (2'R) stereoisomer concerning the epoxide group in the labeling reagent. The results demonstrated that the HL labeled with single Gal-beta1,4-GlcNAc-Epo was functional as a novel N-acetyllactosamine-binding protein, and the second labeling was performed by way of the first-ligand assisted recognition of the second ligand.

X‐ray structural analysis of the ligand‐recognition mechanism in the dual‐affinity labeling of c‐type lysozyme with 2′,3′‐epoxypropyl β‐glycoside of N‐acetyllactosamine

“…The micro-seeding method using native HEWL crystals worked well to give the prisms with the dimensions of 0.3 Â 0.4 Â 0.4 mm, and the diffraction data was collected on an Enraf-Nonius FAST diffractometer using the Cu Ka radiation produced by FR571 generator (40 kV, 50 mA). Crystallization of the doubly labeled Glu102-mutant HL was performed as described previously for the corresponding derivative of wild-type HL (Muraki et al, 1999). Only small prisms with the maximum dimension of 0.1 mm were obtained and the data collection was performed at the device of Photon Factory (Tsukuba, Japan) using the synchrotoron radiation of wavelength of 1.0 Å .…”

“…However, to date, only a few cases, in which the three-dimensional structure of the modified enzyme was determined at high resolution, have been reported with regard to glycosidases (Keitel et al, 1993;Chen et al, 1995;Havukainen et al, 1996). The modified human lysozyme (HL) that uncovered the origin of its carbohydrate-recognition specificity has also constituted part of the limited examples (Muraki et al, 1996(Muraki et al, , 1998(Muraki et al, , 1999(Muraki et al, , 2000a. C-type lysozymes including HL and hen egg-white lysozyme (HEWL) are a family of bacteriolytic enzymes of vertebrate origin.…”

“…Nevertheless, in addition to the 2',3'-epoxypropyl b-glycoside of GlcNAcb1,4GlcNAc (GlcNAcb1,4GlcNAc-Epo), the corresponding derivative of N-acetyllactosamine (Galb1,4Glc-NAc-Epo) has been successfully applied to label the active site of HL (Muraki et al, 1996). To our surprise, not GlcNAcb1,4GlcNAc-Epo but Galb1,4GlcNAc-Epo further reacted with the enzymatically inactive singly modified HL to give the doubly modified HL, and the second labeling was considered to occur by way of first ligand-assisted recognition of the second ligand (Muraki et al, 1999). This phenomenon is interesting from the view point of functional design of a protein, because the result suggested the possibility of creating a novel carbohydrate-binding protein without catalytic activity (a lectin) which shows the recognition specificity different from the major specificity of the original protein using the pre-conjugation of a saccharide moiety.…”

“…This phenomenon is interesting from the view point of functional design of a protein, because the result suggested the possibility of creating a novel carbohydrate-binding protein without catalytic activity (a lectin) which shows the recognition specificity different from the major specificity of the original protein using the pre-conjugation of a saccharide moiety. The crystallographic analysis of the singly modified wild-type HL (Muraki et al, 1996) and the doubly modified wild-type HL (Muraki et al, 1999) revealed the important contribution of the side-chain groups of Asp 102 and Gln 104 in wild-type HL as the protein part to the specific hydrogen-bonding interactions with the ligand part. The previous study also gave a reasonable account on the mechanism of the sequential attachment of a couple of Galb1,4GlcNAc-Epo molecules to the catalytic carboxylate groups of Glu35 and Asp53 in the active site of wild-type HL (Muraki et al, 1999).…”

“…The crystallographic analysis of the singly modified wild-type HL (Muraki et al, 1996) and the doubly modified wild-type HL (Muraki et al, 1999) revealed the important contribution of the side-chain groups of Asp 102 and Gln 104 in wild-type HL as the protein part to the specific hydrogen-bonding interactions with the ligand part. The previous study also gave a reasonable account on the mechanism of the sequential attachment of a couple of Galb1,4GlcNAc-Epo molecules to the catalytic carboxylate groups of Glu35 and Asp53 in the active site of wild-type HL (Muraki et al, 1999). In the present study, the affinity-labeling of two different c-type lysozymes was investigated.…”