Table 2.
CST and CST/UGT chimeric mutations that altered transport and/or substrate recognition. CST and CST/UGT chimeric mutants shown to affect the transport and/or substrate recognition have been summarised. A complete list of all CST and CST/UGT mutants (based on the available literature) assessed including those that had no effect on transport and/or substrate recognition has been included in the supplementary data Table A.
| Mutant | Location | Background | Effect | Experiment | Comments |
|---|---|---|---|---|---|
| C16A | TMD 1 | Site-directed mutagenesis of mouse CST. Generated and expressed in P. pastoris. | Essential for CST substrate specificity | STD NMR spectroscopy | Highly conserved in CSTs, but not present in Mn & Hn UGT [94]. |
| K65A | 2nd loop | Site-directed mutagenesis of mouse CST. Generated and expressed in P. pastoris. | Significant effect on CMP-Neu5Ac recognition. Suggests binding the Sia moiety and not the nucleotide moiety. | STD NMR spectroscopy | Highly conserved in CSTs, but not present in Mn & Hn UGT [94]. |
| Q101H | TMD 3 | Naturally occurring mutation in patient with intellectual disability and bleeding diathesis | 50% reduction in CST activity | Functional assay | [16] |
| L112G & D113G Double mutant |
3rd loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST | EPO/IEF assay a | [101] |
| L136G | 4th loop | MAR-11 cells | Essential for CST activity | EPO/IEF assay a | Highly conserved in CST. UGT conserved counterpart, L160G did not affect UGT [101]. |
| G153A & G154A Double mutant |
TMD 5 | Cotransfection of constructs that express CST into CHO CST-deficient MAR-11 cells. | Slightly reduced activity | EPO/IEF assay a | [99] |
| G153I & G154I Double mutant |
TMD 5 | Cotransfection of constructs that express CST into CHO CST-deficient MAR-11 cells. | Clear reduction in activity | EPO/IEF assay a | [99] |
| G177A & G179A Double mutant |
TMD 6 | Cotransfection of constructs that express CST into CHO CST-deficient MAR-11 cells. | Slightly reduced activity | EPO/IEF assay a | [99] |
| G177I & G179I Double mutant |
TMD 6 | Cotransfection of constructs that express CST into CHO CST-deficient MAR-11 cells. | Clear reduction in activity | EPO/IEF assay a | [99] |
| G189A & G192A Double mutant |
TMD 6 | Cotransfection of constructs that express CST into CHO CST-deficient MAR-11 cells. | Clear reduction in activity | EPO/IEF assay a | [99] |
| G189E | TMD 6 | Site-directed mutagenesis. Expressed in CHO-WT and mutant Lec 2 | Clear reduction in activity | Complementation analysis | Gly189 in highly conserved CST region. Indicates size of aa is critical for activity — not charge [98]. |
| G189Q | TMD 6 | Site-directed mutagenesis. Expressed in CHO-WT and mutant Lec 2 | Clear reduction in activity | Complementation analysis | As above [98]. |
| G189I | TMD 6 | Site-directed mutagenesis. Expressed in CHO-WT and mutant Lec 2 | Clear reduction in activity | Complementation analysis | As above [98]. |
| Y214A | TMD 7 | Site-directed mutagenesis of Mouse CST. Generated and expressed in P. pastoris. | Dramatic effect on CMP-Neu5Ac recognition. Suggests binding of the Sia moiety and not the nucleotide moiety. | STD NMR spectroscopy | Highly conserved in CSTs, but not present in Mn & hUGT [98] |
| Y214G | TMD 7 | hUGT/hCST chimera | Loss of CST activity (retained UGT activity) | Complementation analysis | [70] |
| S216F | TMD 7 | hUGT/hCST chimera | Loss of CST activity (retained UGT activity) | Complementation analysis | [70] |
| 236KGFF239 to 236GGGG239 | 8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST | EPO/IEF assay a | [101] |
| G256I & G257I Double mutants |
TMD 8 | Cotransfection of constructs that express CST into MAR-11 cells. | Clear reduction in activity | EPO/IEF assay a | [99] |
| G256A & G257A Double mutants |
TMD 8 | Cotransfection of constructs that express CST into CST-deficient MAR-11 cells. | Clear reduction in activity | EPO/IEF assay a | [99] |
| 267TDNI270 to 267GGGG270 | 8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST | EPO/IEF assay a | [101] |
| I270G | 8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Partially reduced transport activity | EPO/IEF assay a | [101] |
| I270W | 8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Partially reduced transport activity | EPO/IEF assay a | [101] |
| K272A | 8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST. Essential for CST transport activity | EPO/IEF assay a | [101] |
| K272G | 8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST. Essential for CST transport activity | EPO/IEF assay a | [101] |
| K272H | 8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST. Essential for CST transport activity | EPO/IEF assay a | Changed to His as His is in hGDP-fucose transporter. [101] |
| Deletion of last 4 amino acids IIGV | C-terminal tail | Eliminated the export signals and prevented ER-to-Golgi translocation | [80] | ||
| TMD 2 & 3 | TMD 2 TMD 3 |
hUGT-hCST chimera | Affects the efficiency of CST | [100] | |
| TMD 7 | TMD 7 | hUGT-hCST chimera | Required for substrate specificity | [100] | |
| CST-GFP4 (GFP loop interruption) |
3rd loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST | EPO/IEF assay a | [52,101] |
| CST-GFP8 (GFP loop interruption) |
7th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Complete inactivation of CST | EPO/IEF assay a | [52,101] |
| CST-GFP10 (GFP loop interruption) |
8th loop | CST-GFP12 mutant. Co-transfected with an EPO vector into CHO MAR-11 cells | Partial inactivation of CST | EPO/IEF assay a | [52,101] |
a
EPO/IEF assay: Recombinant human erythropoietin/isoelectric focusing assay.