Anti-Calpain I/II, large subunit monoclonal antibody, clone 2E20B8

The penta-EF-hand (PEF) protein ALG-2 (apoptosis-linked gene 2) has been implicated in several important physiological processes, including endoplasmic reticulum-Golgi vesicular transport and. endosomal biogenesis/transport. ALG-2 was recently shown to harbor a metal ion-binding site with a high affinity for Mg2+ and a low affinity for Ca2+. We herein present the X-ray structure of Mg2+-bound ALG-2des23(wt). Although the C-alpha trace is nearly indistinguishable from that of the Ca2+-free protein, the orientation of the C-terminal helix differs in the two structures. Consistent with that observation, replacement of the +x ligand in EF5, D169, with alanine eliminates high-affinity Mg2+ binding. It also eliminates the low-affinity Ca2+ site and lowers the affinity of the remaining Ca2+-binding sites, EF3 and EF1. The coordination environment in EF5 approaches ideal Mg2+ octahedral geometry. The ligand array, consisting of three carboxylates (+x, +y, +z), a backbone carbonyl (-y), and two water molecules (-x, -z), may offer a recipe for a high-affinity, high-selectivity Mg2+-binding site. Sequence data for other PEF proteins indicate that select calpain large subunits, notably CAPN1 and CAPN8, may also possess a high-affinity Mg2+-binding site. In Mg2+-bound ALG-2, the carbonyl of F188 and the C-terminal carboxylate of V191 interact with the epsilon-ammonium group of K137 in the opposing subunit, suggesting that Mg2+ binding could have an impact on dimerization. Interestingly, EF1 and EF3 are also occupied in the crystal, despite having modest affinity for Mg2+. The results of a calorimetry-based analysis indicate that their Mg2+ binding constants are 2 orders of magnitude lower than that determined for EF5.

Forty loci (16 polymorphic and 24 non-polymorphic) together with 23 cosmids isolated from a chromosome 11-specific library were used to construct a detailed genetic map of 11p13-11q13. The map was constructed by using a panel of 13 somatic cell hybrids that sub-divided this region into 19 intervals, a meiotic mapping panel of 33 multiple endocrine neoplasia type 1 (MEN1) families (134 affected and 269 unaffected members) and a mitotic mapping panel that was used to identify loss of heterozygosity in 38 MEN1-associated tumours. The results defined the most likely order of the 16 loci as being: 11pter-D11S871-(D11S288, D11S149)-11cen-CNTF-PGA-ROM1-D11S480-PYGM-SEA-D11S913-D11S970-D11S97-D11S146-INT2-D11S971-D11S533-11 qter. The meiotic mapping studies indicated that the most likely location of the MEN1 gene was in the interval flanked by PYGM and D11S97, and the results of mitotic mapping suggested a possible location of the MEN1 gene telomeric to SEA. Mapping studies of the gene encoding mu-calpain (CAPN1) located CAPN1 to 11q13 and in the vicinity of the MEN1 locus. However, mutational analysis studies did not detect any germ-line CAPN1 DNA sequence abnormalities in 47 unrelated MEN1 patients and the results therefore exclude CAPN1 as the MEN1 gene. The detailed genetic map that has been constructed of the 11p13-11q13 region should facilitate the construction of a physical map and the identification of candidate genes for disease loci mapped to this region.