DISULFIDE BONDS, N-GLYCOSYLATION AND TRANSMEMBRANE TOPOLOGY OF SKELETAL-MUSCLE TRIADIN

Native triadin is a disulfide linked homopolymer of variable subunit n umber. Two monoclonal antibodies (mAbs), AE8.91 and GE4.90, recognize cytoplasmic regions of triadin between amino acids 110 and 163 and at the C-terminal 34 amino acids, respectively. Triadin in intact triads is largely unaffected by trypsin, while triads whose membrane has been disrupted by hypotonicity or by treatment with the detergent Triton X -100 yield both soluble and membrane bound fragments. Soluble fragment s monitored by mAb GE4.90 appear to be formed sequentially during the course of proteolysis at 28, 16, 10 and 7 kDa in the presence of merca ptoethanol. Higher molecular weight bands are observed under nonreduci ng, conditions. A two-dimensional electrophoresis immunoblot (first no nreducing; second reducing) of the soluble fragments developed with mA b GE4.90 shows the presence of several bands which can be interpreted as containing a dimer formed by a combination of any two of the fragme nts of 16, 10, or 7 kDa present in the digest. MAb AE8.91 does not det ect these fragments. This observation indicates that one of the interm olecular disulfide bonds is formed between the identical domains of tw o triadin molecules at cysteine 671. Immunoblots performed with and wi thout mercaptoethanol of the insoluble fragments using mAb AE8.91 indi cate the presence of a dimer formed between identical domains of two t riadin molecules with a subunit of 60 kDa. This fragment which was not detected with GE4.90 indicates an intermolecular disulfide linkage at cysteine 270. The glycosidase endo F/N-glycosidase F changed the mobi lity of intact triadin in TC/triads and its proteolytic fragments dete cted by mAb GE4.90. It decreased the mass of the 46, 28, and 16 kDa so luble fragments, detected by mAb GE4.90, but not the 10, 7, and 5 kDa species confirming that the asparagine at residue 625 is, glycosidated . The cytoplasmic location of the mAbs together with the conditions of tryptic digestion and the glycosylation site at N-625 provides the fr amework of a model of the transmembrane topology of triadin in which e ach disulfide resides in a separate segment of a membrane-spanning bet a sheet or similar extended structure. An additional beta sheet segmen t and an a helix comprise two more membrane-spanning domains of triadi n giving rise to a model with four membrane transits and extensive cyt oplasmic and luminal regions. The alpha helix segment shares some iden tity with the M2 segment of the ryanodine receptor while the beta shee t segments resemble the sequences of the dihydropyridine receptor whic h are considered to line the pore of the Ca2+ channel. This latter com parison suggests the possibility that triadin may be a channel.