Chlamydomonas reinhardtii has multiple prolyl 4-hydroxylases, one of which is essential for proper cell wall assembly.
Prolyl 4-hydroxylases (P4Hs) catalyze formation of 4-hydroxyproline (4Hyp), which is found in many plant glycoproteins. We cloned and characterized Cr-P4H-1, one of 10 P4H-like Chlamydomonas reinhardtii polypeptides. Recombinant Cr-P4H-1 is a soluble 29-kD monomer that effectively hydroxylated in vitro both poly(l-Pro) and synthetic peptides representing Pro-rich motifs found in the Chlamydomonas cell wall Hyp-rich glycoprotein (HRGP) GP1. Similar Pro-rich repeats that are likely to be Cr-P4H-1 substrates are also present in the cell wall HRGP GP2 and probably GP3. Suppression of the gene encoding Cr-P4H-1 by RNA interference led to a defective cell wall consisting of a loose network of fibrils resembling the inner and outer W1 and W7 layers of the wild-type wall, while the layers forming the dense central triplet were absent. The lack of Cr-P4H-1 most probably affected 4Hyp content of the major HRPGs of the central triplet, GP1, GP2, and GP3. The reduced 4Hyp levels in these HRGPs can also be expected to affect their glycosylation and, thus, the interactive properties and stabilities of their fibrous shafts. Interestingly, our RNA interference data indicate that the nine other Chlamydomonas P4H-like polypeptides could not fully compensate for the lack of Cr-P4H-1 activity and are therefore likely to have different substrate specificities and functions.
Keskiaho
Katriina
K
Collagen Research Unit, Biocenter Oulu and Department of Medical Biochemistry and Molecular Biology, University of Oulu, FIN-90014 Oulu, Finland.
Hieta
Reija
R
Sormunen
Raija
R
Myllyharju
Johanna
J
D020418:Algal Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002473:Cell Wall / Q000201:enzymology*; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018503:Genome, Protozoan; D006909:Hydroxyproline / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D020418:Algal Proteins; D011993:Recombinant Fusion Proteins; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline