1B1, lanes 2 and 3) were both transferred to a PVDF membrane
and submitted to Edman degradation. The first 34 amino acid residues from N-terminal sequencing of the reduced protein were determined to be LGPDIVSPPVCGNELLEVGEECDCGTPENCQNE (Fig. 2) and submitted to BLAST. The 10 first amino acids residues of the non-reduced moojenin obtained by Edman degradation showed the same sequence as the reduced moojenin (data not shown). The primary 17-AAG sequence of the reduced moojenin shared a high degree of identity with proteins of the PIIIb subclass of SVMPs, except for a proline (Pro208) where threonine (Thr208) is observed in other known sequences. This sequence begins at the spacer region in other members of the PIIIb subclass of SVMPs (residue 206 – numbering according to Jararhagin), such as the disintegrins Catrocollastatin-C (Calvete et al., 2000) and Jararhagin-C (Usami et al., 1994), suggesting that the moojenin had undergone autolysis. Subclass PIIIb metalloproteinases can undergo proteolysis/autolysis during secretion or in the Natural Product Library venom to generate disintegrin-like and cysteine-rich domains (DC domain) (Fox and Serrano, 2005). However, no proteinase domain released from the DC domain of a PIIIb metalloproteinease has been isolated intact from snake venom, since they are apparently unstable alone (Shimokawa et al., 1997; Moura-da-Silva
et al., 2003; Fox and Serrano, 2005 and Fox and Serrano, 2008). A spacer region, or linker, separates the M from the DC domain and includes a proteolytic site (Moura-da-Silva et al., 2003; Assakura et al., 2003; Muniz et al., 2008), but the cleavage site is not yet known. It has been observed that proteolytic processing occurs in the spacer domain in some members of each of the P classes Galactosylceramidase (Fox and Serrano, 2005). For example, processed PIIIb catrocollastatin-C (Fig. 2) has a spacer region linked to the disintegrin-like domain just as in reduced moojenin (Fox and Serrano, 2005), while native jararhagin-C (Usami et al., 1994) and ALT-C (Souza et al., 2000) contain only the DC domain. Other members of the PIII class undergo autolysis
under non-physiological conditions in vitro ( Takeya et al., 1993); however, the products of this proteolytic processing are not observed by SDS-PAGE under non-reducing conditions, suggesting these domains are connected by disulfide bonds ( Moura-da-Silva et al., 2003). Moreover, under reducing conditions the DC domain was seen without the M domain, since the latter alone is unstable. Other members of the PIII subclass can be manipulated to undergo in vitro autolysis, but the relevance of this processing in vivo is unclear ( Fox and Serrano, 2005). Under physiological conditions, Moojenin probably maintains both native and processed conformations, since the sequence determined for non-reduced moojenin begins at the spacer region.