Truncated profibrillin of a Marfan patient is of apparent similar size as fibrillin: intracellular retention leads to over-N-glycosylation.
We studied profibrillin-1 (proFib) synthesis and microfibril formation in cultured fibroblasts from an individual with severe Marfan syndrome harboring a premature stop codon (W2756ter) in one FBN1 allele. Rotary shadowing analysis of extracellular matrix produced by these cells revealed the presence of only a very few intact microfibrils which showed marked disorganisation within the interbeaded domains. Metabolic pulse-chase studies identified intracellularly a population of truncated proFib molecules which were secreted more slowly than the normal proFib derived from the normal allele. Culture media contained strikingly reduced amounts of wild-type proFib in comparison to fibrillin (Fib). Our findings imply that (1) the truncated proFib is secreted and disturbs microfibril assembly; (2) the mutation is probably close to a putative cleavage site in the proFib C terminus necessary for the conversion of proFib to Fib; (3) the truncated proFib is over-N-glycosylated due to intracellular retention rather than incomplete cleavage of proFib with persistence of N-glycosylated sites; (4) not all potential N-glycosylation sites in proFib seem to be normally used, since we could produce over-N-glycosylated proFib in normal cells by brefeldin A mediated intracellular captivation and subsequent appearance of over-glycosylated Fib in culture medium upon removal of the compound. It is conceivable that post-translational over-modification might be important for modulating the phenotype of FBN1 mutations in Marfan syndrome.
Raghunath
M
M
Dept. of Paediatrics, University of Zürich, Switzerland.
Kielty
C M
CM
Steinmann
B
B
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D020126:Brefeldin A; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology; D016326:Extracellular Matrix Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D000071838:Fibrillin-1; D000071837:Fibrillins; D005347:Fibroblasts; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D008382:Marfan Syndrome / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D054392:Adipokines
D020126:Brefeldin A; D003517:Cyclopentanes; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; D014415:Tunicamycin; C000605722:FBN1 protein, human; D054392:Adipokines