4.2 E XPRESSION AND SUBCELLULAR DISTRIBUTION IN MAMMALIAN CELLS
4.2.9 Effect of glycosylation on protein transport and cell surface expression
4.2.9.2 Expression of PLKC in glycosylation deficient CHO/Lec cells
In order to further dissect the possible impact of glycosylation on the subcellular distribution and cell surface transport of PLKC, a cell system defective of correct glycosylation was established. CHO/Lec1 cells, missing the acetylglucosaminyl-transferase I, are no more capable of transfering sialic acids, galactose and N-acetylglucosamine (GalNAc) residues to the mannose tail (Puthalakath et al., 1996).
CMP-siliac acid transporters are not expressed by CHO/Lec2 cells (Deutscher et al., 1984), affecting N-glycosylation as well as O-glycosylation: carbohydrate branches lack sialic acids, which are normally coupled in the trans golgi compartment.
Finally CHO/Lec8 cells do not exhibit efficient UDP-galactose transporters and thereby they are unable to link galactose and sialic acid sugars to the polypeptide (Deutscher and Hirschberg, 1986;Oelmann et al., 2001).
All three cell types were stably transfected with PLKC-YFP, generating the new cell lines CHO/Lec1-PLKC-YFP, CHO/Lec2-PLKC-YFP and CHO/Lec8-PLKC-YFP.
PLKC-YFP expressed in wild-type CHO cells (CHO-PLKC-YFP) was used as a control.
First, biochemical analysis of PLKC expression in the established glycosylation defective cell lines and CHO-PLKC-YFP were carried out using immunoprecipitation of PLKC-YFP with mAb anti-GFP after 8 hours pulse with [35S] methionine (Figure 24). To identify the band corresponding to the mannose rich form of PLKC, samples were finally subjected to treatment with endo H.
The first two lanes show PLKC expressed by wild-type CHO cells, exhibiting the same pattern already observed in MDCK-II cells (cp. 4.2.7 Biochemical analysis of PLKC), i.e. a band of around 170 kDa corresponding to the endo H-sensitive PLKCh, (lane 2, ~ 145 kDa) and the complex glycosylated form (PLKCc, approximately 210 kDa), which is not affected by endo H digestion.
In contrast, PLKC expressed by CHO/Lec1 cells is represented by only a single band at a size of around 160 kDa, which is completely sensitive to endo H digestion. This phenotype is due to the defective activity of N-acetylglucosaminyl-transferase I in CHO/Lec1 cells, where newly synthesized proteins are prevented from being further processed by addition of N-acetylglucosamine (GlcNAc), galactose or sialic acid to the mannose rich core. Therefore, the created glyco-polypeptide is fully sensitive towards endo H and no resistant complex glycosylated isoform is generated.
Proteins isolated from CHO/Lec2-PLKC and CHO/Lec8-PLKC cells both show a complex glycosylated form (lane 5 - 8). But whereas CHO/Lec2-PLKC cells (lane 5 and 6) exhibit nearly the same glycosylation pattern as the wild type, in CHO/Lec8 cells (lane 7 and 8) the ratio between PLKCc and PLKCh is changed in favour of the latter one. Furthermore, in CHO/Lec8 cells, the complex band is shifted down (~
20kDa) and not as expanded as the one of the wild type (lane 1 and 2) or as it is in case of CHO/Lec2 cells. These effects are the consequence of impaired N-glycosylation in the cis-golgi of CHO/lec8 cells: defects in the UDP-galactose transporters lead to a blocked addition of sialic acids to the premature glycopolypeptide and therefore, the complex form is diminished.
CHO/Lec2 cells only lack N- and O-linked sialic acids due to a malfunction of CMP-siliac acid transporters and therefore the difference to CHO-wild type cells is not as significant as it is in case of the other mutants.
These oberservations lead to the conclusion that the O- as well as the N-gylcosylation mainly consist of N-acetylglucosamine (GlcNAc) and galactose residues, whereas sialic acids, added in the trans-Golgi compartment are represented to a much less extent.
The difference of approximately 10 kDa between both, the complex and the mannose rich forms of PLKC expressed in the glycosylation mutants on the one hand (~ 200 kDa and ~ 160 kDa respectively) and the one generated in “wild type” CHO cells on the other hand (~ 210 kDa and ~ 170 kDa) is caused by a different processing of the precursor protein in the ER. In CHO/Lec cells only five mannose residues are linked to the two GlcNAc linked to asparagine residues of the core protein, whereas in “wild type” CHO cells nine mannose molecules are attached.
CHO/K1- CHO/Lec1- CHO/Lec2- CHO/Lec8-PLKC CHO/Lec8-PLKC CHO/Lec8-PLKC CHO/Lec8-PLKC endo H - + - + - + - +
PLKCc PLKCh
210kDa 170kDa
145kDa
Figure 24 Expression of PLKC in glycosylation-mutant CHO cells
PLKC-YFP was stably transfected into CHO/Lec1, CHO/Lec2 and CHO/Lec8 cells. The clones and CHO-PLKC-YFP cells, which served as a control, were biosynthetically labeled for 8 hours with [35S]
methionine, and PLKC-YFP was immunoprecipitated from total cell lysates using mAb anti-GFP.
Beads were treated with Endo H (+) or not (-).
4.2.9.2.2 Analysis of subcellular distribution by confocal laser microscopy The intracellular localization of the chimeric PLKC-YFP in transfected CHO/Lec cells was determined using confocal laser microscopy (Figure 25).
apical lateral basal
30µm 30µm 30µm
apical lateral basal
30µm 30µm 30µm
CHO/Lec1
apical lateral basal
25µm
25µm 25µm
CHO/Lec2
apical lateral basal
25µm
25µm 25µm
CHO/Lec8
Figure 25 Confocal analysis of PLKC in glycosylation mutant CHO cells
In all three glycosylation defective cell types, PLKC is predominantly localized at intercellular cell contact sites (blue arrows) and on the cell surface (white arrow) although a partial staining for vesicular structures was also observed (red arrows).
Although CHO cells are non-polarizing cells, the Lec mutant cells exhibit a cell shape and expression pattern of PLKC on the cell surface which resembles strikingly some epithelial cell lines like MDCK-II. This is even more convincing when XZ-sections of confluent monolayer are scanned (Figure 26). PLKC is sorted to the “apical”
membrane and to the lateral cell-cell contacts nearly to the same extent like it was shown in MDCK-II cells (cp. 4.2.2 Redistribution of PLKC). So we can address here a kind of directed apical and lateral transport, which is obviously not effected by impaired glycosylation of the protocadherin. Because the data acquired by inhibitors of glycosylation (cp. 4.2.9 Effect of glycosylation on protein transport and cell surface expression) and confocal experiments using the CHO/Lec cells were absolutely convincing, no cell surface biotinylation or trypsin assays were performed with the glycosylation mutant cell lines to show that PLKC-YFP indeed reaches the cell surface.
XZ-section scan
Figure 26 XZ-section scan of glycosylation defective cells expressing PLKC-YFP CHO/Lec2-PLKC-YFP cells were grown until five days after confluence and analyzed using the XZ-section scanning method in laser microscopy. Similar results were obtained using CHO/Lec1-PLKC-YFP or CHO/Lec8-PLKC-CHO/Lec1-PLKC-YFP cells.