Extended Data Fig. 3
From: Staphylococcal phosphatidylglycerol antigens activate human T cells via CD1a
(a) LysylPG was treated at the indicated pH at 25 °C overnight (16 hr) or 37 °C for 1 hr, followed by lipid extraction and HPLC–MS analysis. The PG and lysylPG were quantified as percentage of the sum of both lipids, based on the curve fitting of external standards. (b) Isoelectric focusing gel showing the migration pattern of human CD1a carrying heterogenous mixture of lipids derived from the mammalian expression system (CD1a-endo) upon incubation with lysylPG or PG at low (citrate pH 5.5) or high (tris pH 8) pH. Presence of negatively charged lipids (for example PG) in the cleft of CD1a shifts the PI of the protein towards electronegative values (CD1a−1). Degradation of lysylPG to PG at high pH results in migration pattern identical to CD1a-PG. (c) CD1a proteins treated with lysylPG (pH 5.5 or pH 8) described in (b) were extracted and the eluted lipids, lysylPG and PG were analyzed by HPLC–MS. (d) Isoelectric focusing gel showing distinct migration pattern of CD1a incubated with CHAPS, PG or lysylPG based on the pI of the protein. LysylPG-loaded CD1a was further incubated overnight in MMT buffer at different pH to assess the stability of the lysylated headgroup. The lack of band corresponding to more negatively charged species of CD1a (CD1a-1) suggests that lysylPG is stable once bound to CD1a.
