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. 2006 Nov 28;45(47):14223-31.
doi: 10.1021/bi061677n.

Proteins in load-bearing junctions: the histidine-rich metal-binding protein of mussel byssus

Hua Zhao  1 , J Herbert Waite
Affiliations

Proteins in load-bearing junctions: the histidine-rich metal-binding protein of mussel byssus

Hua Zhao et al. Biochemistry. .

Abstract

Building complex load-bearing scaffolds depends on effective ways of joining functionally different biomacromolecules. The junction between collagen fibers and foamlike adhesive plaques in mussel byssus is robust despite the strikingly dissimilar connected structures. mcfp-4, the matrix protein from this junction, and its presecreted form from the foot tissue of Mytilus californianus were isolated and characterized. mcfp-4 has a mass of approximately 93 kDa as determined by MALDI-TOF mass spectrometry. Its composition is dominated by histidine (22 mol %), but levels of lysine, arginine, and aspartate are also significant. A small amount of 3,4-dihydroxyphenyl-l-alanine (2 mol %) can be detected by amino acid analysis and redox cycling assays. The cDNA-deduced sequence of mcfp-4 reveals multiple variants with highly repetitive internal structures, including approximately 36 tandemly repeated His-rich decapeptides (e.g., HVHTHRVLHK) in the N-terminal half and 16 somewhat more degenerate aspartate-rich undecapeptides (e.g., DDHVNDIAQTA) in the C-terminal half. Incubation of a synthetic peptide based on the His-rich decapeptide with Fe3+, Co2+, Ni2+, Zn2+, and Cu2+ indicates that only Cu is strongly bound. MALDI-TOF mass spectrometry of the peptide modified with diethyl pyrocarbonate before and after Cu binding suggests that histidine residues dominate Cu binding. In contrast, the aspartate-rich undecapeptides preferentially bind Ca2+. mcfp-4 is strategically positioned to function as a macromolecular bifunctional linker by using metal ions to couple its own His-rich domains to the His-rich termini of the preCOLs. Ca2+ may mediate coupling of the C-terminus to other calcium-binding plaque proteins.

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Figures

FIGURE 1
FIGURE 1
Schematic drawing of the mussel byssus illustrating a close-up of the junction between the thread and plaque portions and the extensive interface between the splayed collagen fibrils and foam proteins of the plaque (right). Gel electrophoresis (acid urea-PAGE) of the proteins extracted from three microdissected portions of freshly secreted byssal threads (left) [(A) footprint region, (B) junction, and (C) distal thread]. The gels have been stained for Dopa with glycinate and nitroblue terazolium (NBT). Identification of known protein families is according to previous studies (9-16). The boxed band is mcfp-4, and the double-headed arrow marks the position of an internal calibrant, the α chain of type I collagen.
FIGURE 2
FIGURE 2
mcfp-4 from adhesive plaques. (A) Immobilized metal affinity chromatography (IMAC) of Mcfp-4 with a chelating column charged with Zn2+. Protein was eluted with a stepped increase to pH 3. (B) Gel filtration chromatography on Shodex 803 of the eluted IMAC-binding fractions. His-rich fractions are denoted with a bracket. (C) Acid urea-polyacrylamide gel electrophoresis of the key steps in mcfp-4 purification: crude soluble plaque extract from plaques stained with Coomassie blue R-250 (lane 1), IMAC column binding fractions stained in parallel for protein with CBR-250 (lane 2), Dopa with NBT in glycinate (lane 3), histidine with Pauly’s reagent (lane 4), and Shodex-purified mcfp-4 (arrow) stained with CBR-250 (lane 5). The double-headed arrow marks an internal calibrant, the α chain of type I collagen.
FIGURE 3
FIGURE 3
mcfp-4 from mussel feet. (A) Acid urea-polyacrylamide gel electrophoresis of key steps in the purification of foot-derived mcfp-4. Guanidine-extracted and borate-dialyzed (pH 8.0) soluble protein stained with CBR-250 (lane 1), stained for Dopa with NBT (lane 2), and stained for histidine with Pauly’s reagent (lane 3). The remaining lanes (lanes 4-8) contained fractions under the bracketed peak of the reverse phase C8 HPLC column (B) stained with CRB-250. The double-headed arrow marks an internal calibrant, the α chain of type I collagen.
FIGURE 4
FIGURE 4
MALDI-TOF mass spectrum of purified mcfp-4 from both mussel feet and plaques. Peaks from right to left were [M + H]+ and [M + 2H]2+. Delayed extraction (200 ns) in positive ion mode with an accelerating voltage of 25 000 V, a grid voltage at 93%, and a guide wire voltage at 0.1%. The spectrum represents an average of 256 scans.
FIGURE 5
FIGURE 5
Complete deduced sequence of mcfp-4, variant 1. The schematic shows the domains of mcfp-4. The italicized portion is the signal peptide; underlined sequences denote those determined directly by Edman degradation. Variant 2 differs because it has five additional histidine-rich decapeptides.
FIGURE 6
FIGURE 6
MALDI-TOF mass spectra of the synthetic His-rich decapeptide dimer and its metal adducts. The peptide (HGHVHTHRVLHKHVHKHRVL), excerpted from the histidine-rich N-terminal domain, was incubated with a 10-fold molar excess of Fe3+, Ni2+, Zn2+, Co2+, and Cu2+ in 25 mM N-ethylmorpholine and 30 mM KCl buffer (pH 7.4).
FIGURE 7
FIGURE 7
MALDI-TOF mass spectra of DEPC modification of the synthetic His-rich decapeptide before (A) and after (B) Cu2+ incubation. Integers over the peaks represent the calculated number of carbethoxylations. Note that all eight of the His residues and two other functionalities appear to be monocarbethoxylated DEPC (A, i), which prevents any Cu2+ binding that can be detected by MALDI-TOF (A, ii). Conversely, Cu2+ binding prior to DEPC exposure abolishes monocarbethoxylation by any peptide functionality (B, i and ii).
FIGURE 8
FIGURE 8
Calcium binding behavior of the synthetic peptide from the C-terminal undecapeptides of mcfp-4 assessed by MALDI-TOF MS.
FIGURE 9
FIGURE 9
Proposed model of mcfp-4’s role in joining the histidine-rich domains of the splayed preCOLs and other calcium binding proteins present in the adhesive foam. The participation of mcfp-2 is predicated on its abundance and the known calcium binding activity of its EGF domains. The involvement of the phosphorylated variants of mcfp-5 and -6 cannot be precluded.
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