Original Paper Abstract: Allergenicity of plant glycoproteins in humans may prevent the use of plants as production factories for pharmaceu- tically important proteins. The major difference between plant and mammalian N-glycans is the presence of xylosyl and a1,3- fucosyl residues in the former. In a first step towards ªhumaniza- tionº of the N-glycosylation pathway in the moss Physcomitrella patens, which could be an excellent system for industrial produc- tion of therapeutic proteins, we isolated the cDNAs and genes for N-acetylglucosaminyltransferase I (GNTI), a 1,3-fucosyltrans- ferase, and b1,2-xylosyltransferase. Sequence analysis revealed that all three proteins are homologous to their counterparts from higher plants, however, the conservation of the primary structure was only 35 ± 45 %. The gene encoding the key enzyme of the pathway, gntI, was disrupted in P. patens by homologous recombination. Although the mutation of this gene in mouse or A. thaliana led to a significantly altered pattern of N-glycans, the glycosylation pattern in the gntI knockouts did not differ from that in wild-type moss and was identical to that in higher plants. Protein secretion, analysed in assays with recombinant human VEGF 121 protein, was not affected in the knockouts. We conclude from our findings that the N-glycosylation pathway in P. patens is identically organized to that in higher plants. However, P. pat- ens probably possesses more than one isoform of GNTI which complicates a straightforward knockout. Therefore, and since complex type structures appear more desirable than oligoman- nosidic N-glycans, future modifications of the pathway should target a1,3-fucosyltransferase and/or b1,2-xylosyltransferase. Key words: Gene knockout, N-acetylglucosaminyltransferase I, a1,3-fucosyltransferase, b 1,2-xylosyltransferase, N-glycosyla- tion, Physcomitrella patens. Abbreviations: GNTI: N-acetylglucosaminyltransferase I a1,3-Fuc-T: a1,3-fucosyltransferase b1,2-Xyl-T: b1,2-xylosyltransferase GlcNAc, Gn: N-acetylglucosaminyl residue Man, M: mannose residue GF: Lewis A determinant X: xylosyl residue F: fucosyl residue HPLC: high-performance liquid chromatography MALDI-TOF-MS: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry VEGF: human vascular endothelial growth factor Footnote: The nucleotide sequences reported in this paper were submitted to the GenBank  /EBI Data Bank with accession numbers AJ429143 (GNTI), AJ429145 (a1,3-Fuc-T), and AJ429144 (b1,2-Xyl-T). Introduction Manysecretedproteins,aswellasintegralmembraneproteins of the secretory system, are glycoproteins, i.e., they are modi- fied by glycans (oligosaccharides) that are N-linked to aspara- gineorO-linkedtoserine,threonine,orhydroxyproline.N-gly- cosylationisresponsibleforcorrectfoldingandstabilityofthe proteins,preventionofproteindegradation,proteinconforma- tion and recognition, solubility of proteins, their secretion to the extracellular space, and their biological activity. As in other eukaryotes, N-glycosylation of plant proteins starts in the endoplasmic reticulum (ER) by the transfer of an oligosaccharide precursor onto specific Asn residues. Further processing of plant N-linked glycans occurs along the secre- tory pathway as the glycoprotein moves from the ER through the Golgi apparatus to its final destination. Glycosidases and glycosyltransferases located in the ER and the Golgi succes- sively modify the oligosaccharide precursor to high man- nose-type N-glycans and then into complex N-glycans. In the first step of the Golgi-localized processing, a-mannosi- dase I removes one to four mannose residues and converts Man 9 GlcNAc 2 to Man 5 GlcNAc 2 (Fig. 1) (Sturm et al.,1987). The biosynthesis of complex N-glycans starts with the addition of N-acetylglucosamine (GlcNAc) to the mannose branch, cata- lysed by N-acetylglucosaminyltransferase I (GNTI), yielding GlcNAcMan 5 GlcNAc 2 (Johnson and Chrispeels, 1987). Two ad- ditional mannose residues are removed by a-mannosidase II andanotherGlcNAcresidueistransferredbyN-acetylglucosa- minyltransferase II (GNTII) to the end of the second mannose branch.Afterthisreaction,plantandanimalglycosylationsep- arate: in plants, the N-glycans are further modified by a1,3- fucosylation and b1,2-xylosylation of the core Man 3 GlcNAc 2 (Johnson and Chrispeels,1987; Tezuka et al.,1992; Zeng et al., N-Glycosylation in the Moss Physcomitrella patens is Organized Similarly to that in Higher Plants A. Koprivova 1 , F. Altmann 2 , G. Gorr 3 , S. Kopriva 4 , R. Reski 1 , and E. L. Decker 1 1 University of Freiburg, Plant Biotechnology, 79104 Freiburg, Germany 2 Institut für Chemie, Universität für Bodenkultur, Muthgasse 18,1190 Vienna, Austria 3 Greenovation Biotech GmbH, Bötzinger Straûe 29b, 79111 Freiburg, Germany 4 University of Freiburg, Department of Tree Physiology, Georges-Köhler-Allee 053, 79085 Freiburg, Germany Received: July 16,2003; Accepted: November11, 2003 PlantBiology5(2003):582±591 GeorgThiemeVerlagStuttgart´NewYork ISSN1435-8603´DOI10.1055/s-2003-44721 582