A HYDROXYPROLINE-CONTAINING CLASS-IV CHITINASE OF SUGAR-BEET IS GLYCOSYLATED WITH XYLOSE

Two acidic chitinase isoforms, SP1 and SP2, have been purified to homogeneity from leaves of sugar beet (Beta vulgaris) infected with Cercospora beticola. SP1 and SP2 are extracellular proteins with an apparent molecular mass of 35 kDa and an approximate pi of 4.2. Since the only major difference was slightly diverging M(r)'s, only the SP2 chitinase was further characterized. Partial amino acid sequence data for SP2 was used to generate a polymerase chain reaction (PCR) clone employed for the isolation of a cDNA clone encoding SP2. SP2 exhibits significant structural identity with the class TV chitinases from sugar beet, rapeseed, bean and maize, but differs from the other members of this class in having a longer hinge region, comprising 22 amino acid residues, with a repeated 'TTP' motif. Western blotting analyses, using antibody raised against SP2, demonstrated an induction of SP protein during infection with C. beticola. The induction was very local, with high protein accumulation found close to the infection site only. Amino acid compositional analysis of SP2 revealed that five out of fourteen prolines are hydroxylated. No glucosamine or galactosamine residues are present. Evidence was obtained that SP2 is glycosylated with a limited number (less than or equal to 7) of xylose residues: (1) SP2 was stained with the periodic acid-Schiff (PAS) reagent, (2) electrospray mass spectrometry on SP2 gave a series of M(r)'s with a consistent increase between two molecular masses of 132 Da, (3) SP2 was recognized by an antibody specific for beta-1,4-D-xylopyranose. The vacuolar class I chitinases A and B in tobacco have recently been shown to comprise a new class of hydroxyproline-containing proteins (Sticher et al., Science 257 (1992) 655-657). The SP2 chitinase differs from these in being glycosylated and, thus, represents a novel type of hydroxyproline-containing glycoproteins in plants.