Carbon Starvation Increases Endoglycosidase Activities and Production of "Unconjugated N-Glycans" in Silene alba Cell-Suspension Cultures

Abstract

We previously reported the occurrence of oligomannosides and xylomannosides corresponding to unconjugated N-glycans (UNGs) in the medium of a white campion (Silene alba) cell suspension.Attention has been focused on these oligosaccharides since it was shown that they confer biological activities in plants. In an attempt to elucidate the origin of these oligosaccharides, we studied two endoglycosidase activities, putative enzymes involved in their formation. The previously described peptide-N'-(N-acetyl-glucosaminyl) asparagine amidase activity and the endo-N-acetyl-8-Dglucosaminidase activity described in this paper were both quantified in white campion cells during the culture cycle with variable initial concentrations of sucrose. The lower the sucrose supply, the higher the two activities. Furthermore, endoglycosidase activities were greatly enhanced after the disappearance of sugar from the medium. The production of UNCs in the culture medium rose correlatively. These data strongly suggest that the production of UNCs in our white campion cell-suspension system is due to the increase of these endoglycosidase activities, which reach their highest levels of activity during conditions of carbon starvation.Xylomannosides Man3(Xyl)GlcNAc(Fuc)GlcNAc and GlcNAcMan~(Xyl)GlcNAc(Fuc)GlcNAc and the oligomannoside ManaGlcNAc have been isolated from the extracellular suspension-culture medium of white campion (Silene alba) (Priem et al., 1990b; Lhemould et al., 1992). The importance of these UNGs was recently emphasized (Priem et al., 1994). Several glycoproteins conjugated to these oligosaccharides have been reported (Kamerling, 1991). The excretion and accumulation of the UNGs in the medium at the end of the culture period could be explained by glycoprotein breakdown. When deprived of SUC, cells consume starch, protein, and lipid reserves (Joumet et al., 1986). We have recently demonstrated the presence of peptide-N4-(N-acetyl-glucosaminyl) Asn amidase, named PNGase Se, in the cell extract of white campion (Lhemould et al., 1992). This enzyme hydrolyzes the glycosylamine linkage. Oligomannosides and xylomannosides are effective substrates for PNGase Se only when they are carried by small peptides and not by proteins or glycoasparagines (S. Lhemould, Y . Karamanos, H. Morvan, unpublished data).In this paper we show the presence of endo-N-acetyl-8-D-* Corresponding author; fax 33-55-45-73-86. 779glucosaminidase, named ENGase Se, in cell extracts of white campion, which could explain the occurrence of the oligomannoside described earlier (Priem et al., 1990b), and we have also evaluated the influence of exogenous Suc concentration on the level of endoglycosidase activities. MATERIALS A N D METHODS Cell CultureWhite campion (Silene alba [Miller] E.H.L. Krause) suspension culture was obtained by the Dubois and Bourriquet (1973) procedure. Subcultures were produced in 500-mL flasks with 200 mL of medium as previously described (Morvan, 1982), except that the growth period was extended to 30 d. Suc (10, 20, or 40 g/L) was i...

Novel Molecular Markers for Late Phases of the Growth Cycle of Arabidopsis thaliana Cell-Suspension Cultures Are Expressed during Organ Senescence

“…A number of reports have described the expression of glycosyl hydrolases in nutrient-starved cell cultures, e.g. a-amylases from rice (Chen et al, 1994;Sheu et al, 1994), P-glucosidase from Brassica (Malboobi and Lefebvre, 1995), or N-acetyl-p-glucosaminidase from Silene (Lhernoud et al, 1994). It is well known that certain hydrolytic activities may be generated by the release of the corresponding enzymes from various cellular storage compartments.…”

Regulation of De-N-Glycosylation Enzymes in Germinating Radish Seeds

“…Using Silene alba cellsuspension cultures, a temporal delay has been shown to occur between the increase in PNGase and ENGase activities in the cells and the appearance of UNGs in the culture medium (Lhernould et al, 1994). This phenomenon was correlated with carbon starvation.…”

Free N-linked oligosaccharide chains: Formation and degradation

“…The dose-dependent influence of UNGs on both growth and senescence of plant cells has been observed [83,86,94]. It is also known that the amount of UNGs changes during fruit ripening or cell culture [94,95], suggesting the correlation of OS metabolism and observed biological phenomena. There are two deglycosylating enzymes so far known in plants; plant-specific acid PNGase [84,[95][96][97][98][99][100][101][102][103][104][105][106][107] and cytoplsmic ENGase [95,105,[108][109][110][111][112][113][114].…”

“…It appears that the acidic PNGase is responsible for the release of complex type glycans, while the ENGase is involved in generation of high mannose-type, Gn1 species. These enzyme activities also appear to be regulated in a developmental stage-dependent manner [95,105,107]. 5 Example structures of (a) high mannose type and (b) complex type UNG, plant-type free OS.…”