Extended Data Fig. 1: Phosphoserines mediate protein binding to alum and influence immune response.
From: Engineered immunogen binding to alum adjuvant enhances humoral immunity
(a) Unmodified cytochrome-C protein or an equivalent concentration of cytochrome-C conjugated with a single pSer linker containing 1, 2, or 4 phosphoserine groups was mixed with 100 µg/mL Alhydrogel in 10 mM MOPS buffer. Alum was separated by centrifugation and bound protein was detected using absorbance at 410 nm compared to a standard curve of known protein concentration. (b) Peptide linkers of equivalent molecular size were prepared, composed of an azide functional group linked to either 4 phosphoserines, or 3 serines and 1 phosphoserine. These linkers were coupled to DBCO-modified phycoerythrin (PE) via copper-free click chemistry at a range of total linkers per protein. (c) pSer/Ser-conjugated PE (5 µg/mL) with varying numbers of linkers per protein was mixed with alhydrogel (50 µg/mL) for 30 min, followed by addition of mouse serum (final concentration 10% vol/vol) for 24 hr. Protein bound to alum after this incubation was assessed by fluorescence spectroscopy. (n=3 samples/group). Data represents mean ± SD. (d) PAGE gel of eOD protein reacted with pSer4-eOD. PNGase treatment was used with eOD to remove glycans prior to running gel. Representative gel from two experiments. Gel image is cropped. (e) eOD proteins (10 µg/mL) conjugated with a single 4-residue serine linker, or pSer linkers containing 1-8 phosphoserines, were incubated with AlHydrogel (100 µg/mL) for 30 min in TBS at 25 °C, followed by measurement of bound protein by fluorescence. (n=3 samples/group). Center lines represent mean and error bars represent SD. (f) Day 63 sera from BALB/c mice (n=5 animals/group) immunized with eOD (black) or pSer4-eOD (red) were tested for binding to pSer tags. Plates were coated with pSer4-cytochrome c to quantify the pSer-specific response. Data represents mean ± SD. (g, h) Germinal center B cells isolated from the inquinal lymph nodes of BALB/c mice (n= 10 animals/group, pooled from 2 experiments) 9 days after immunization with eOD variants and Al-Hydrogel. Cell counts are provided for GC B cells (g) and eOD-specific GC B cells (h). Center lines represent mean and error bars represent SD. Statistical tests were performed using one-way ANOVA with Tukey’s post test.
