title: Quantitative transcriptomic profiling of branching in a glycosphingolipid biosynthetic pathway.
abstract: Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCerSyn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected.
authors.affiliation: Laboratory of Membrane Biochemistry and Biophysics, Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto 606-8501, Japan. htakema@pharm.kyoto-u.ac.jp
authors.forename: Hiromu
authors.initials: H
authors.lastname: Takematsu
authors.forename: Harumi
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Yuko
authors.initials: Y
authors.lastname: Naito-Matsui
authors.forename: Reiko
authors.initials: R
authors.lastname: Fujinawa
authors.forename: Kouji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Okuno
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Mamoru
authors.initials: M
authors.lastname: Kyogashima
authors.forename: Reiji
authors.initials: R
authors.lastname: Kannagi
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Kozutsumi
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000378:metabolism; D002451:Cell Compartmentation; D002460:Cell Line; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006028:Glycosphingolipids / Q000096:biosynthesis*; D006801:Humans; D012190:Retroviridae / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D006028:Glycosphingolipids; D015061:1,4-alpha-Glucan Branching Enzyme