THE 5TH EPIDERMAL GROWTH-FACTOR-LIKE DOMAIN OF THROMBOMODULIN DOES NOT HAVE AN EPIDERMAL GROWTH-FACTOR-LIKE DISULFIDE BONDING PATTERN

The disulfide bonding pattern of the fourth and fifth epidermal growth factor (EGF)-like domains within the smallest active fragment of thro mbomodulin have been determined, In previous work, this fragment was e xpressed and purified to homogeneity, and its cofactor activity, as me asured by k(cat) for thrombin activation of protein C, was the same as that for full-length thrombomodulin. CNBr cleavage at the single meth ionine in the connecting region between the domains and subsequent deg lycosylation yielded the individual EGF-like domains. The disulfide bo nds were mapped by partial reduction with tris(2-carboxyethyl)phosphin e according to the method of Gray [Gray, W. R. (1993) Protein Sci. 2, 1732-1748], which provides unambiguous results. The disulfide bonding pattern of the fourth EGF-like domain was (1-3, 2-4, 5-6), which is th e same as that found previously in EGF and in a synthetic version of t he fourth EGF-like domain. Surprisingly, the disulfide bonding pattern of the fifth domain was (1-2, 3-4, 5-6), which is unlike that found i n EGF or in any other EGF-like domain analyzed so far. This result is in line with an earlier observation that the (1-2, 3-4, 5-6) isomer bo und to thrombin more tightly than the EGF-like (1-3, 2-4, 5-6) isomer. The observation that not all EGF-like domains have an EGF-like disulf ide bonding pattern reveals an additional element of diversity in the structure of EGF-like domains.