A REPETITIVE PROLINE-RICH PROTEIN FROM THE GYMNOSPERM DOUGLAS-FIR IS A HYDROXYPROLINE-RICH GLYCOPROTEIN

Intact cell elution of suspension cultures derived from Douglas fir, P seudotsuga menziesii (Mirbel) Franco, yielded two extensin monomers, t he first hydroxyproline-rich glycoproteins (HRGPs) to be isolated from a gymnosperm. These HRGPs resolved on Superose-6 gel filtration. The smaller monomer was compositionally similar to angiosperm extensins li ke tomato P1. The larger monomer had a simple composition reminiscent of repetitive proline-rich proteins (RPRPs) from soybean cell walls an d contained proline, hydroxyproline, and sugar; hence designated a pro line-hydroxyproline-rich glycoprotein (PHRGP). The simple composition of the PHRGP implied a periodic structure which was confirmed by the s imple chymotryptic map and 45-residue partial sequence of the major pr oline-hydroxyproline-rich glycoprotein chymotryptide 5: -Hyp-Val-Tyr-L ys-IIe-Pro-Pro(Hyp)-Val-IIe-Lys-Pro. Proline-hydroxyproline-rich glyco protein chymotryptide 5 contained an 18-residue tandem repeat devoid o f tetra(hydroxy)-proline or serine; it also contained two instances of the five-residue motif Hyp-Hyp-Val-Tyr-Lys and five of the general Pr o-Pro-X-X-Lys motif, thereby establishing its homology with typical an giosperm RPRPs and extensins from tomato, petunia, carrot, tobacco, su gar beet, and Phaseolus. Unlike the nonglycosylated soybean RPRP, the highly purified Douglas fir PHRGP was lightly glycosylated, confirmed by a quantitative hydroxyproline glycoside profile, indicating that ex tensins can range from highly glycosylated hydroxyproline to little or no glycosylated hydroxyproline. Comparison of extensin sequence data strongly indicates that a major determinant of hydroxyproline glycosyl ation specificity is hydroxyproline contiguity: extensins with tetrahy droxyproline blocks are very highly arabinosylated (> 90% hydroxyproli ne glycosylated), tri- and dihydroxyproline are less so, and single hy droxyproline residues perhaps not at all. Despite high yields of exten sins eluted from intact cells, the Douglas fir cell wall itself was hy droxyproline poor yet remarkably rich in protein (> 20%), again emphas izing the existence of other structural cell wall proteins that are ne ither HRGPs nor glycine-rich proteins.