Poster: Cell Walls

Abs # 1248: Synthetic genes for the elucidation of p3-type extensin crosslinking amino acids

Presenter:	Held, Michael A., extensin3@yahoo.com
Authors	Held, Michael A. (A)   Leykam, Joseph F. (B)   Kieliszewski, Marcia J. (A)
Affiliations:	(A): Department of Chemistry and Biochemistry, Ohio University (B): Department of Biochemistry, Michigan State University

P3 extensins are hydroxyproline-rich glycoproteins (HRGP’s) in the primary wall that function in cell growth and development, as well as wound and disease responses. Secreted to the wall presumably as soluble monomeric precursors, P3 extensins are covalently crosslinked into the wall. Although genes encoding P3 extensin have been isolated, as have fragments of P3-type extensins from wall digests, the intact monomeric precursor has never been isolated and characterized, presumably due to very rapid wall insolublization. To circumvent this problem and to examine the requirements for crosslinking, we have designed and expressed in tobacco BY2 cells, synthetic genes encoding the P3-type extensin repeat unit (SOOOOSOSOOOOYYYK)_n (O=Hyp) with enhanced green fluorescent protein (EGFP) as the reporter protein. We tested the influence of Y and K on P3 intermolecular crosslinking by synthesizing Y to F and K to L variants of the P3 putative crosslinking motif YYYK. The P3-EGFP fusion proteins and variants were hydroxylated, contained isodityrosine (IDT), and were arabinosylated as predicted by earlier work with endogenous P3 peptides. Assays catalyzed by extensin peroxidase indicated tyrosine, but not lysine is required for intermolecular crosslinking.