title: Calcium binding, hydroxylation, and glycosylation of the precursor epidermal growth factor-like domains of fibrillin-1, the Marfan gene protein.
abstract: The extracellular matrix protein fibrillin-1 is a major component of elastic microfibrils, which are complex assemblies of several proteins and are found in most connective tissues, frequently associated with elastin. Fibrillin-1 contains 43 precursor epidermal growth factor-like (pEGF) domains that have a consensus sequence for calcium binding. The calcium binding potential of a fibrillin-1 pepsin fragment (PF2) was quantitatively analyzed using microvolume equilibrium dialysis. Peptide sequence data and pepsin fragment size determination indicate that PF2 contains seven pEGF domains, each with the calcium binding consensus sequence. Scatchard plot analysis of the calcium binding data shows that PF2 has six to seven high affinity binding sites with a Kd = 250 microM at pH 7.5. There is a second overlapping consensus sequence in the pEGF domains for beta-hydroxylation of a specific Asp/Asn residue. Five partially hydroxylated Asn residues have been identified by protein sequence analysis of fibrillin-1 fragments. This is the first demonstration of this modification in a connective tissue protein. The calcium binding consensus sequence also contains a conserved Ser residue with an apparently novel modification, which causes the Ser residue to behave like an Asp residue during protein sequencing. Marfan syndrome, a heritable disorder of connective tissue, is known to be associated with mutations in the FBN1 gene. Most of these mutations have been found in pEGF domains, frequently substituting Cys for another amino acid, destroying the pEGF motif secondary structure along with its calcium binding potential. Other mutations cause the substitution of single amino acids in the calcium binding consensus sequence, which could affect calcium binding but also the hydroxylation of Asp/Asn residues or the modification of Ser residues.
authors.affiliation: Research Department, Shriners Hospital for Crippled Children, Portland, Oregon.
authors.forename: R W
authors.initials: RW
authors.lastname: Glanville
authors.forename: R Q
authors.initials: RQ
authors.lastname: Qian
authors.forename: D W
authors.initials: DW
authors.lastname: McClure
authors.forename: C L
authors.initials: CL
authors.lastname: Maslen
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry; D002240:Carbohydrate Sequence; D016384:Consensus Sequence; D003956:Dialysis; D004815:Epidermal Growth Factor / Q000737:chemistry; D016326:Extracellular Matrix Proteins / Q000737:chemistry / Q000378:metabolism; D000071838:Fibrillin-1; D000071837:Fibrillins; D006031:Glycosylation; D006801:Humans; D006900:Hydroxylation; D008382:Marfan Syndrome / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D054392:Adipokines
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D004815:Epidermal Growth Factor; D016326:Extracellular Matrix Proteins; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D010434:Pepsin A; D010446:Peptide Fragments; D011498:Protein Precursors; C000605722:FBN1 protein, human; D054392:Adipokines; C042652:epidermal growth factor precursor