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.

Psychosine-triggered endomitosis is modulated by membrane sphingolipids through regulation of phosphoinositide 4,5-bisphosphate production at the cleavage furrow

“…This value was called the p sychosine-mediated p loidy i ndex n umber (PPIN). When the six–cell line profile of PPIN was expressed as a web graph ( Figure 1C ), a similarity was found in the pattern with that of cell surface GM1 expression level, measured with the cholera toxin B subunit (CTxB), as in a previous study with the same set of cell lines ( Takematsu et al. , 2011 ).…”

Blood Groups in Infection and Host Susceptibility

“…Globo-GSLs are also sensitive to mutations and agents affecting Golgi function. Mutations in FAPP2 or treatment of cells with brefeldin leads to a loss of globo-GSL, with increased GM3 and LacCer levels (355,356). Glycosyltransferases necessary for the synthesis of early precursors (GlcCer, LacCer, and GM3) reside in the endoplasmic reticulum and cis-Golgi network, whereas glycosyltransferases necessary for globo-GSL synthesis reside in the trans-Golgi network (357).…”

“…Glycosyltransferases necessary for the synthesis of early precursors (GlcCer, LacCer, and GM3) reside in the endoplasmic reticulum and cis-Golgi network, whereas glycosyltransferases necessary for globo-GSL synthesis reside in the trans-Golgi network (357). There is evidence that ␣4GalT1 and LacCer synthase form an intra-Golgi complex to efficiently synthesize globo-GSLs (356).…”

Sphingolipid Metabolism and Interorganellar Transport: Localization of Sphingolipid Enzymes and Lipid Transfer Proteins

“…(iii) IEM analysis demonstrated that A4GalT is enriched in the TGN . A4GalT can bind to B4GalT6 (it is unknown whether A4GalT can also bind to B4GalT5) , suggesting that A4GalT may recruit LacCer synthases to the TGN.…”