The EC domains of human fibrinogen420 contain calcium binding sites but lack polymerization pockets.

The extended (E) isoform unique to Fibrinogen420 (Fib420) is distinguished from the conventional chain of Fibrinogen340 by the presence of an additional 236-residue carboxyl terminus globular domain (EC). A recombinant form of EC (rEC), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography. Purified rEC appears to be predominantly intact, as judged by N-terminal sequence analysis, mass spectral analysis of the C-terminal cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies. Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in E. Analysis of CNBr digestion fragments confirms that two disulfide bridges exist at cysteine pairs E613/644 and E780/793. In the presence of 5 mmol/L EDTA, rEC is highly susceptible to plasmic degradation, but Ca2+ (5 mmol/L) renders rEC resistant. No protective effect from plasmic degradation was conferred to rEC by the peptides GPRPamide or GHRP, nor did rEC bind to a GPR peptide column. These results suggest that the EC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated in the gammaC domain, we predict that the EC calcium binding site involves residues E772-778: DADQWEE.

Distribution of desmosomal proteins in F9 embryonal carcinoma cells and epithelial cell derivatives.

In diverse epithelia, cytoskeletal keratin intermediate filaments (IFs) associated with the cytoplasmic face of intercellular junctional desmosomes. The processes underlying desmosome formation and keratin IF interactions remain unclear. We have examined F9 embryonal carcinoma (EC) cell differentiation as a model for embryonic development of epithelial surface desmosomes. As determined by immunofluorescence microscopy and biochemical protein techniques, F9 EC cells, which lack surface desmosomes and keratin IFs, express the desmosomal proteins desmoplakins I and II (DP I/II), desmoglein I (DG I) and plakoglobin (PK). DP I/II are present at low level and are relatively soluble in buffer containing Triton X-100. Immunofluorescence localizes DP I/II to the juxtanuclear, centrosomal region. Species of DG I and PK are detected in both the Triton X-100-soluble and -insoluble protein fractions. DG I appears dispersed throughout the cell while PK resides at cell-cell boundaries. In epithelial cell cultures induced by retinoic acid (RA) treatment, each of the desmosomal proteins is organized into punctate desmosome-like structures with the appearance of simple epithelial K8/K18 IFs. The steady-state levels of DP I/II and PK increase with a partitioning of the majority of the desmosomal components into the insoluble fraction. In epithelial cells which lack distinct surface desmosomes, an intracellular association of keratin bundles with DP I/II is observed, suggesting that keratin filaments may facilitate the translocation of these desmosomal components to the cell surface. Parietal endoderm-like cells, derived by treatment with RA and dibutyryl cAMP, are analogous to F9 EC cells in that the cells express desmosomal components and do not display surface desmosomes. Moreover, K8 and K18 do not form distinct filaments, and the protein and RNA levels of K8 are low relative to epithelial cells induced by RA alone. The F9 system appears to be a relevant model for studies of desmosome assembly and the potential interactions of desmosomal proteins and keratin IFs in embryonic epithelial cell types.