Glycosylated Polyproline II Rods with Kinks as a Structural Motif in Plant Hydroxyproline-Rich Glycoproteins ² Patrick J. Ferris, § Jeffrey P. Woessner, §,| Sabine Waffenschmidt, ⊥ Sven Kilz, ⊥ Jutta Drees, ⊥ and Ursula W. Goodenough* ,§ Department of Biology, Washington UniVersity, St. Louis, Missouri 63130, and Institute fu¨ r Biochemie, UniVersitat zu Ko¨ ln, Ko¨ ln, Germany ReceiVed October 10, 2000; ReVised Manuscript ReceiVed December 20, 2000 ABSTRACT: Hydroxyproline-rich glycoproteins (HRGPs) are the major proteinaceous components of higher plant walls and the predominant components of the cell wall of the green alga Chlamydomonas reinhardtii. The GP1 protein, an HRGP of the C. reinhardtii wall, is shown to adopt a polyproline II helical configuration and to carry a complex array of arabinogalactoside residues, many branched, which are necessary to stabilize the helical conformation. The deduced GP1 amino acid sequence displays two Ser- Pro-rich domains, one with a repeating (SP) x motif and the other with a repeating (PPSPX) x motif. A second cloned gene a2 also carries the PPSPX repeat, defining a novel gene family in this lineage. The SP-repeat domains of GP1 form a 100-nm shaft with a flexible kink 28 nm from the head. The gp1 gene encodes a PPPPPRPPFPANTPM sequence at the calculated kink position, generating the proposal that this insert interrupts the PPII helix, with the resultant kink exposing amino acids necessary for GP1 to bind to partner molecules. It is proposed that similar kinks in the higher plant HRGPs called extensins may play a comparable role in wall assembly. Hydroxyproline-rich glycoproteins (HRGPs), 1 first identi- fied in dicots but since found in monocots and green algae (1-3; reviewed in refs 4-7), are the major proteinaceous components of the plant cell wall and often become co- valently cross-linked into large meshworks (8, 9). Biochemi- cal and molecular studies of these proteins, which have been assigned to several subgroups (4, 5), have documented commonalitiessrepeated motifs dominated by (hydroxy)- proline and serine, a polyproline II helical conformation, and arabinosyl/galactosyl side chainssthat have led Kieliszewski and Lamport (10) to propose that they derive from an ancient gene family. The extensin subgroup of the HRGP family includes proteins that are uniformly fibrous and apparently devoted to meshwork formation, but several members of the sub- group, called chimeric extensins (10), display both fibrous and globular domains, the globular portions presumably playing some additional role. These include several solona- ceous lectins, where the hydroxyproline-rich fibrous domains are thought to anchor the protein to the wall and the globular domains to mediate sugar-binding activity (reviewed in refs 4 and 5). Chimeric HRGPs are also expressed in the reproductive tissues of monocots (11, 12) and dicots (13- 15), where their functions have not yet been elucidated. Algal HRGPs have been largely studied in Chlamydomo- nas and VolVox (reviewed in refs 16 and 17), organisms that construct their vegetative and zygotic cell walls exclusively from HRGPs. In addition to an “inner wall” of covalently cross-linked HRGPs reminiscent of the higher plant mesh- works (18), they possess as well an “outer wall” of HRGPs that coassemble into crystalline arrays. The arrays can be solubilized in chaotropic salts, and they reassemble when the salts are removed by dialysis (18-23). These outer-wall HRGPs represent excellent subjects for molecular and morphological studies on HRGP interactions. Most of the volvocine HRGPs thus far characterized prove to be chimeric proteins, with globular “heads” and hydroxy- proline-rich “shafts.” Two classes of proline-rich motifs (when discussing HRGP-encoding genes, the residues are referred to as proline, most of which are posttranslationally converted to hydroxyproline) have been identified to date in Chlamydomonas and VolVox: in the first, the prolines are contiguous; in the second, the prolines alternate with some other amino acid, most often serine. We report here a new Pro-rich motif, identified in two cloned genes of Chlamy- domonas reinhardtii, that is characterized by regular repeats of PPSPX, thereby generating sequences that carry both contiguous and noncontiguous prolines. One chimeric PPSPX gene, called a2, is expressed exclusively in gametes, where the function of its protein product has not yet been elucidated. A second chimeric PPSPX gene, called gp1, encodes the GP1 protein which coassembles with two other HRGPs (GP2 and GP3) to form the salt-soluble outer wall of C. reinhardtii ² Supported by NIH Grant GM-26150, NSF Grant MCB-9904667, and Fonds der Chemischen Industrie. * Corresponding author. Tel: 314-935-6836. Fax: 314-935-5125. E-mail: ursula@biosgi.wustl.edu. § Washington University. | Current address: Paradigm Genetics, Research Triangle Park, NC 27709. ⊥ Universitat zu Ko¨ln. 1 Abbreviation: HRGP, hydroxyproline-rich glycoprotein. 2978 Biochemistry 2001, 40, 2978-2987 10.1021/bi0023605 CCC: $20.00 © 2001 American Chemical Society Published on Web 02/10/2001