Oligomerization of the sensory and motor neuron-derived factor prevents protein O-glycosylation.
The sensory and motor neuron-derived factor (SMDF) is a neuregulin that promotes Schwann cell proliferation and differentiation. Hence, understanding axon myelination is important to unveil the mechanisms involved in SMDF biogenesis, membrane delivery, and compartmentalization. SMDF is a type II membrane protein expressed as two distinct polypeptides of approximately 40 and 83 kDa. Whether the 83-kDa polypeptide results from posttranslational modifications of the protein monomers or protein dimerization remains unknown. Here we have addressed this question and shown that the 83-kDa polypeptide is an O-glycosylated form of the protein. Deletion of the N-terminal domain fully abrogates the SMDF O-glycosylation, indicating that incorporation of O-glycans occurs in the intracellular domain of the protein. Notably, O-glycosylated forms are excluded from partitioning into lipid raft microdomains. In addition, we found that heterologously expressed SMDF monomers interact in intact living cells as evidenced from fluorescence resonance energy transfer of cyan fluorescent protein/yellow fluorescent protein.SMDF fusion proteins. A stepwise deletion approach demonstrated that SMDF self-association is primarily determined by its transmembrane segment. Notably, biochemical analysis revealed that SMDF multimers are exclusively composed of the 40-kDa polypeptide. Collectively, these findings indicate that the 40-kDa form corresponds to unmodified SMDF, which may be present as multimers, whereas the 83-kDa polypeptide is a monomeric O-glycosylated form of the protein. Furthermore, our observations imply a role for oligomerization as a potential modulator of the distribution in membrane domains and O-glycosylation of the protein.
Cabedo
Hugo
H
Instituto de Neurociencias, Universidad Miguel Hernández-Consejo Superior de Investigaciones Científicas, Alicante 03550, Spain. hugo.cabedo@umh.es
Carteron
Christelle
C
Ferrer-Montiel
Antonio
A
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D017353:Gene Deletion; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D007163:Immunosorbent Techniques; D008164:Luminescent Proteins / Q000235:genetics; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D012583:Schwann Cells / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014162:Transfection
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins