Identification of Cell Surface Straight Chain Poly-N-Acetyl-Lactosamine Bearing Protein Ligands for VH4-34–Encoded Natural IgM Antibodies

(A) Changes in SC-PNAL on Nalm-6 cells following treatment with agents that alter protein glycosylation. Mean and SD of three independent experiments is shown. Statistical significance was determined with Student t test using Prism software. (B) Changes in SC-PNAL expression after methanol treatment. Mean channel fluorescence (MCF) on methanol-fixed cells compared with control cells is shown as a percentage. Mean and SD of three independent experiments is shown. Expression of CD77 was tested on Daudi, and CD17 and SC-PNAL expression was tested on Nalm-6. (C) Effect of lipid glycosylation inhibitors on SC-PNAL expression. MCF of electronically gated viable propidium iodide–negative cells is shown. Expression of CD77 and cholera-toxin ligand GM1 (monosialo-tetrahexosylganglioside) was tested on Daudi, and CD17 and SC-PNAL expression was tested on Nalm-6. Expression of SC-PNAL was tested by mAb216. Binding was also tested in OCI-Ly8 treated with the two inhibitors showing decrease in CD17 but not SC-PNAL expression (data not shown).

SC-PNAL is associated with DIF in detergent-treated OCI-Ly8 cells. (A) Change in mean channel fluorescence of detergent-treated cells compared with untreated cells is shown as a percentage. Mean and SD of five independent experiments is shown. Partitioning of SC-PNAL to DIF was also demonstrated in Nalm-6 and Reh (data not shown). (B) Intact cytoskeleton is necessary for partitioning of SC-PNAL to DIF. Mean and SD of three independent experiments is shown.

Immunoprecipitated proteins by mAb 216 identified by TL. The positively stained gel band (solid rectangle) analyzed by Nano-ESI-MS yielded 238 proteins, of which the top 28 proteins identified as membrane proteins or membrane-associated proteins by scaffold GO annotation are shown in Table III. The Western blot analysis of immunoprecipitated material with TL was performed five times independently with identical staining. The Nano-ESI-MS analysis was performed once.

Western blot analysis of immunoprecipitated proteins by VH4-34–encoded Abs by the standard IP. (A) Anti-CD147 mAb F-5 and anti-mouse IgG-HRP. (B) Anti-CD147 mAb HIM-6 and anti-mouse IgG-HRP. (C) Histogram of SC-PNAL and CD147 expression on the Nalm-6 and Peer. Both cell lines have high expression of CD147. The VH4-34–encoded IgM Abs, 216, Y2K, 55.5, Z2D2, and Z21 have independent CDR3 and light chains. All are encoded by germline VH4-34 gene. The dotted line indicates lanes spliced from multiple blots.

IP of CD147 by VH4-34 mAbs is glycosylation specific. Nalm-6 (A) or Peer (B) membrane extract was immunoprecipitated with mAb 216 or isotype control (lanes 1, 2) or anti-CD147 mAb, 8D6 on Protein G (lane 3). Western blot performed with anti-CD147 mAb, F-5, and anti-mouse IgG-HRP. The two bands seen in the secondary alone are the mouse H and L chain of mAb 8D6. The dotted line indicates lanes spliced from multiple blots. Only HG-CD147 was immunoprecipitated by other VH4-34 mAbs 55.5 and Y2K from Nalm-6, OCI-Ly8, and Reh (data not shown).

(A) CD147 and CD98 proteins immunoprecipitated by mAb 216 are immunoprecipitated again by TL. (B) CD147-CD98 complex is immunoprecipitated from other human B cell lines, Reh, and OCI-Ly8. The dotted line indicates reordering of lanes from a single blot. ND, not done.