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

Two acidic chitinase isoforms, SP1 and SP2, have been purified to homo geneity from leaves of sugar beet (Beta vulgaris) infected with Cercos pora 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 chitinas e 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 significa nt structural identity with the class TV chitinases from sugar beet, r apeseed, bean and maize, but differs from the other members of this cl ass in having a longer hinge region, comprising 22 amino acid residues , with a repeated 'TTP' motif. Western blotting analyses, using antibo dy raised against SP2, demonstrated an induction of SP protein during infection with C. beticola. The induction was very local, with high pr otein accumulation found close to the infection site only. Amino acid compositional analysis of SP2 revealed that five out of fourteen proli nes are hydroxylated. No glucosamine or galactosamine residues are pre sent. Evidence was obtained that SP2 is glycosylated with a limited nu mber (less than or equal to 7) of xylose residues: (1) SP2 was stained with the periodic acid-Schiff (PAS) reagent, (2) electrospray mass sp ectrometry on SP2 gave a series of M(r)'s with a consistent increase b etween two molecular masses of 132 Da, (3) SP2 was recognized by an an tibody specific for beta-1,4-D-xylopyranose. The vacuolar class I chit inases A and B in tobacco have recently been shown to comprise a new c lass of hydroxyproline-containing proteins (Sticher et al., Science 25 7 (1992) 655-657). The SP2 chitinase differs from these in being glyco sylated and, thus, represents a novel type of hydroxyproline-containin g glycoproteins in plants.