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

Native triadin is a disulfide linked homopolymer of variable subunit number. 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 fragments 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 mercaptoethanol. Higher molecular weight bands are observed under nonreducing, conditions. A two-dimensional electrophoresis immunoblot (first nonreducing; second reducing) of the soluble fragments developed with mAb 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 fragments of 16, 10, or 7 kDa present in the digest. MAb AE8.91 does not detect these fragments. This observation indicates that one of the intermolecular disulfide bonds is formed between the identical domains of two triadin molecules at cysteine 671. Immunoblots performed with and without mercaptoethanol of the insoluble fragments using mAb AE8.91 indicate the presence of a dimer formed between identical domains of two triadin 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 mobility of intact triadin in TC/triads and its proteolytic fragments detected by mAb GE4.90. It decreased the mass of the 46, 28, and 16 kDa soluble 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 framework of a model of the transmembrane topology of triadin in which each disulfide resides in a separate segment of a membrane-spanning beta sheet or similar extended structure. An additional beta sheet segment and an a helix comprise two more membrane-spanning domains of triadin giving rise to a model with four membrane transits and extensive cytoplasmic and luminal regions. The alpha helix segment shares some identity with the M2 segment of the ryanodine receptor while the beta sheet segments resemble the sequences of the dihydropyridine receptor which are considered to line the pore of the Ca2+ channel. This latter comparison suggests the possibility that triadin may be a channel.