CALCIUM BINDING, HYDROXYLATION, AND GLYCOSYLATION OF THE PRECURSOR EPIDERMAL GROWTH FACTOR-LIKE DOMAINS OF FIBRILLIN-1, THE MARFAN GENE PROTEIN

The extracellular matrix protein fibrillin-1 is a major component of e lastic microfibrils, which are complex assemblies of several proteins and are found in most connective tissues, frequently associated with e lastin. Fibrillin-1 contains 43 precursor epidermal growth factor-like (pEGF) domains that have a consensus sequence for calcium binding. Th e calcium binding potential of a fibrillin-1 pepsin fragment (PF2) was quantitatively analyzed using microvolume equilibrium dialysis. Pepti de sequence data and pepsin fragment size determination indicate that PF2 contains seven pEGF domains, each with the calcium binding consens us sequence. Scatchard plot analysis of the calcium binding data shows that PF2 has six to seven high affinity binding sites with a K-d = 25 0 mu M 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 prot ein sequence analysis of fibrillin-1 fragments. This is the first demo nstration of this modification in a connective tissue protein. The cal cium 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 fo und in pEGF domains, frequently substituting Cys for another amino aci d, destroying the pEGF motif secondary structure along with its calciu m binding potential. Other mutations cause the substitution of single amino acids in the calcium binding consensus sequence, which could aff ect calcium binding but also the hydroxylation of Asp/Asn residues or the modification of Ser residues.