Amino‐acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale

Abstract

The ginger proteases (GP-I and GP-II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P 2 position. The complete amino-acid sequence of GP-II, a glycoprotein containing 221 amino acids, and about 98% that of GP-I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine±histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is < 50%. GP-II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision-induced dissociation MS showed that both sites were occupied by the glycans (Man) 3 (Xyl) 1 (Fuc) 1 (GlcNAc) 2 and (Man) 3 (Xyl) 1 (Fuc) 1 (GlcNAc) 3 , in a ratio of < 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man136(Man133) (Xyl132)Man134GlcNAc134(Fuc133)GlcNAc for the major form, with an additional N-acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.Keywords: amino-acid sequence; cysteine protease; ginger; glycoprotein; glycosylation site; proline peptidase; proteinase; mass spectrometry. Some 20 families of peptidases dependent on a cysteine residue at the active center are known [1]. The most prominent of these is the papain family, which includes peptidases with a wide variety of activities, including endopeptidases with broad specificity (e.g. papain) and narrow specificity (glycyl endopeptidase), amino peptidases, and a dipeptidyl peptidase. Enzymes of the papain family are found in a wide variety of life forms: baculovirus, eubacteria, yeast, probably all protozoa, plants and animals. Papain homologs are generally either lysosomal (vacuolar) or secreted proteins. In plants, they are found in the vacuoles, but also extracellularly, as in the latex of papaya or fig. Plant cysteine proteinases include papain from papaya (Carica papaya), bromelain from pineapple stem and actinidin from the Chinese gooseberry or kiwi fruit (Actinidia chinensis) [1].Ginger proteases from ginger rhizome, Zingiber officinale, were first reported by Ichikawa et al.[2] in 1973. Two proteases, designated GP-I and GP-II were isolated by chromatography on DEAE-cellulose and tentatively classified as cysteine proteases based on their inhibition by thiol reagents and their relative molecular masses, estimated at < 22 500 by gel filtration. More recently, these proteases were separated as mercuribenzoate derivatives into three fractions by IEF [3]. It has been reported [4] that these proteases have both proteinase and collagenase activities; ginger root extracts have also been tested as a meat tenderizer [5,6].Rec...

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