Table 3.
Summary of effects on enzymatic activity and glycosylation for all PAM variants that were functionally tested.
| PAM variant | PHM activity (% WT) | PAL activity (% WT) | PNGaseF (N-sugars) |
Neuraminidase (sialic acid) |
|---|---|---|---|---|
| WT | 100 ± 2 | 100 ± 3 | yes | yes |
| Val27Ile | 79 ± 6 | 103 ± 15 | (yes) | (yes) |
| Phe31Tyr | 76 ± 16 | 84 ± 1 | (yes) | (yes) |
| Val49Leu | 80 ± 26 | 93 ± 1 | (yes) | (yes) |
| Arg80Gln | 75 ± 6 | 87 ± 9 | (yes) | (yes) |
| Thr189Ile † | 6 ± 3** | 23 ± 5** | yes | no |
| His240Tyr | 111 ± 24 | 98 ± 22 | yes | no |
| Val244Ala | 113 ± 32 | 80 ± 10 | (yes) | (yes) |
| Pro412Leu | 81 ± 32 | 85 ± 28 | (yes) | (yes) |
| Ile452Val | 112 ± 45 | 113 ± 30 | (yes) | (yes) |
| Glu491Asp | 133 ± 75 | 138 ± 55 | (yes) | (yes) |
| His529Arg † | 41 ± 13 | 7 ± 1** | n.a. | n.a. |
| Ser539Trp ‡ | 49 ± 7** | 0 ± 1** | yes | no |
| Gly552Arg | 41 ± 6** | 6 ± 2** | yes | no |
| Asp563Gly | 44 ± 8** | 14 ± 3** | yes | no |
| Gly641Arg | 104 ± 0 | 116 ± 10 | (yes) | (yes) |
| Arg703Gln | 92 ± 17 | 0 ± 1** | yes | yes |
| Phe759Ser | 110 ± 25 | 12 ± 3** | yes | no |
| His778fs | 23 ± 3** | 0 ± 1** | n.a. | n.a. |
| Gly796Glu † | 55 ± 8 | 4 ± 1** | (yes) | (yes) |
Values are presented as mean ± SEM. “yes” and “no” refer to the presence and absence, respectively, of N-linked sugars or sialic acid in the variants tested for glycosylation. “(yes)” was given when the effect on glycosylation was based only on SDS-PAGE profile.
n.a., not assessed.
†Engineered inactive mutants.
‡We prepared two independent plasmid constructs and verified by sequencing that they were correct. The p.Ser539Trp variant disrupted the normal trafficking of membrane PAM; when present in the soluble protein, it may inhibit its secretion (15).
** p < 0.0001.