Identification of the phosphorylation site responsible for the hyperphosphorylation of paratarg-7. (A) Immunoelectrophoretic focusing after endopeptidase treatment of paratarg-7. IEF separation followed by immunodetection with anti-paratarg-7 resulted in an additional band of the patient's LCL lysate after trypsin treatment (C) and a modified migration of the chymotryptic fragment (B). The differences in migration of the immunopositive fragments in lanes B and C indicate a different electric charge of the fragments that are the result of the additional phosphorylation. Lane A indicates LCL lysate; lane B, LCL lysate incubated with chymotrypsin; and lane C, LCL lysate incubated with trypsin. (B) Identification of the phosphorylation site responsible for the hyperphosphorylation of paratarg-7 in patients with paratarg-7– specific paraproteins. A phosphorylation site located between aa 1 to 25 is responsible for the hyperphosphorylation of paratarg-7. Lane c indicates recombinant fragment without complementation as control; lane –, recombinant fragment incubated with an enzyme mix from LCLs of a healthy donor carrying wild-type paratarg-7; and lane +, recombinant fragment incubated with an enzyme mix derived from LCLs of a patient carrying hyperphosphorylated paratarg-7. (C) Mutagenization of paratarg-7 fragments containing aa 1 to 62. (D) Isoelectric focusing of the respective fragments shown in panel C. Only the fragment containing Ser17 shows the additional band representing the hyperphosphorylated peptide after complementation with an enzyme mix derived from a carrier of hyperphosphorylated paratarg-7. Lane a indicates expression of the mutagenized fragment as control; lane b, incubation with a native lysate of LCLs from a healthy donor carrying wild-type paratarg-7; and lane c, incubation with a native lysate of LCLs derived from a patient carrying hyperphosphorylated paratarg-7.