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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Fan
H
H
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33136, USA.
Brandt
N R
NR
Caswell
A H
AH
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D002462:Cell Membrane / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D000939:Epitopes / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D008565:Membrane Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018482:Muscle, Skeletal / Q000737:chemistry*; D017830:Octoxynol / Q000494:pharmacology; D017433:Protein Structure, Secondary; D011817:Rabbits; D016415:Sequence Alignment; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002352:Carrier Proteins; D004220:Disulfides; D000939:Epitopes; D008565:Membrane Proteins; D009124:Muscle Proteins; C070048:triadin; D017830:Octoxynol; D006026:Glycoside Hydrolases; D014357:Trypsin