Deletion of extracellular cadherin repeats

The extracellular domain of PLKC consists of seven so called “cadherin repeats” (cp.

Introduction), which are highly conserved amino acid sequences expressed in all variants of the cadherin superfamily. It has been extensively shown that these motifs play a crucial role in protein transport, folding and function. One more aim of this study was therefore to determine the effect of deletion of individual cadherin repeats on the mentioned protein properties. Thus, seven plasmids were constrcuted, in which the single cadherin repeats were repleaced by a spacer of six glycines.

Therefore the pEYFP-PLKC construct was used as a template for “loop-out”

mutagenesis PCR reactions. The strategy is based on the ability of the primers of annealing to both a complementary sequence upstream and a complementary sequence downstream the target repeat, which was substituted with a nucleotide sequence coding the six-glycine spacer (for more detailed information see 3.2.2.9 Construction of domain deletion mutants). The constructs obtained were termed pEYFP-PLKC∆1-7 numbered from the 5’ end and following the open reading frame.

Correct deletion of each repeat was verified by restriction analysis using the enzymes listed in

Table 6, which resulted in the itemized fragments.

construct enzyme(s) used resulting fragments

(control) in bp resulting fragment in bp

125

Table 6 Restriction analysis of cadherin-motif-deletion constructs

For a first screening of possible effects caused by deletion of different cadherin-motifs, the mutants were transfected into COS-1 cells and their intracellular distribution was scanned by confocal analysis.

Only the PLKC∆1 mutant showed a significant difference with respect to the wild type (Figure 39). Here, the modified protein, bearing the deletion of the first cadherin motif, localized immediately after the signal sequence, is retained in the ER and can reach neither the cell surface nor the Golgi compartment. Compartment localization was confirmed using an ER marker and results were compared with the distribution of wild type PLKC (cp. Figure 8). A nearly complete co-localization with the ER marker was found, demonstrating that PLKC∆1 is indeed blocked in the ER and not capable to be further processed.

The other constructs (PLKC∆2-∆7) showed more or less the same distribution pattern as the full-length protein (cp. Figure 8).

11.3µm PLKC∆1-YFP

ER-dsRed

merge

Figure 39 Expression of PLKC∆1 in COS-1 cells

COS-1 cells were co-transfected with PLKC∆1-YFP and ER-dsRed as a marker for the endoplasmic reticulum. 48 hours after transfection subcellular distribution was analysed using confocal microscopy.

Additionally, expression of the PLKC mutants was evaluated also biochemically by immunoprecipitation (Figure 40). Although the relative proportion between the two glycoforms is different, both a mannose rich and a complex glycosylated form are synthesized in the case of PLKC∆2-5 and PLKC∆7, similarly to the wild type PLKC.

However, the mannose rich form in the mutants is represented as a band that is much stronger than the mature protein. These findings reflect a slower processing of the proteins out of the ER through the Golgi and to the cell surface. In fact, proteins could be reasonable retained longer in the ER because of an impaired folding, due to the absence of one single motif of cadherin repeats causing alterations in the secondary structure.

In accordance with the confocal data, PLKC∆1 is exclusively expressed as mannose rich precursor protein (lane 1). It represents the unprocessed polypeptide precursor retained completely in the ER and therefore fully sensitive to endo H (lane 2), due to the absence of complex glycans.

The PLKC∆6 mutant, displaying a distribution very similar to the wild type in a steady state situation (see the fluorescence analyses), is however transported much slower

also than the other mutants, when analysed after biosynthetic labelling. Compared with the wild type (lanes 15 and 16), the mannose rich form is predominant, whereas the complex form results in a very faint band (lane 11 and 12).

PLKC ∆1 ∆2 ∆3 ∆4 ∆5 ∆6 ∆7 wt endo H - + - + - + - + - + - + - + - +

210kDa 170kDa

Figure 40 Immunoprecipitation of cadherin motif deletion mutants in COS-1 cells COS-1 cells were transiently transfected with either wild type PLKC (lane 15 and 16) or the clones PLKC∆1-7 lacking single cadherin repeats. 40 hours later, cells were radioactively labeled with [³⁵S]

methionine for 6 hours. After cell lyses, PLKC-mutants were immunoprecipitated using mAb anti-GFP.

Beads were treated with endo H (+) or not (-).

To explore the behaviour of the mutant PLKC∆6 in more detail, CHO and MDCK-2 cells were stably transfected with the construct PLKC∆6-YFP. The transport kinetic and the intracellular distribution of the mutant PLKC∆6 were analysed by biochemical assays and laser confocal microscopy, respectively.

In pulse chase experiments, cells were labelled either for 15 minutes ( Figure 41A) or for 2.5 hours (

Figure 41B) and chased for different time intervals. In both cell systems, i.e. MDCK and CHO cells (not shown), the PLKC∆6 protein is obviously processed very slow. In fact, the complex glycosylated form appears not after 18 hours of chase, in comparison with the 15-30 minutes observed in the presence of the wild type. The band relative to the mannose rich form persists also after 24 hours of chase.

chase (min) 0 30 60 120 240 360

pulse 15 min

endo H - + - + - + - + - + - +

PLKC_h 155kDa

130kDa

A

chase (h) 0 6 12 18 24

pulse 2,5h

PLKC_c PLKC_h

155kDa 130kDa 195kDa

B

Figure 41 Pulse chase analysis of PLKC∆6-YFP in MDCK-II cells

MDCK- PLKC ∆6-YFP cells were labeled for 15 minutes (A) or 2,5 hours (B) with [³⁵S] methionine and subsequently chased with non radioactive culture medium for the indicated times.

Similar results were obtained using CHO-PLKC∆6-YFP cells in pulse-chase experiments. Next, the intracellular distribution of PLKC∆6 in MDCK-II and CHO cells were examined also by confocal laser microscopy. The results are in line with the first screening experiments performed in COS-1 cells, because the subcellular distribution is in both cell types (MDCK-II and CHO-cells) not significantly altered in comparison to wild type PLKC (data not shown). In MDCK-II cells a distinct amount of protein is still present on the apical cell surface and there is also a strong labelling of the intercellular cell-contact compartment.

Taken together, it can be stated that the first cadherin repeat plays a crucial role in protein transport and a deletion of this domain is linked with transport incompetence and retainment in the ER. PLKC∆6 is transport competent and able to reach the apical cell surface, but the trafficking of the protein is significantly decreased.

Therefore, a deletion of this repeat causes some modification either by deletion of additional sorting signals or by some alteration of protein folding and tertiary structure.

4.4.2 Role of the PDZ-binding site in transport and subcellular