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Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel alpha-helix that links strands 4 and 5 of its beta-sandwich structure. Replacement of the alpha-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the alpha-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 alpha-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.

Although mimics of human tumor antigens are effective immunogens to overcome host unresponsiveness to the nominal antigen, the structural basis of this mimicry remains poorly defined. Therefore, in this study we have characterized the structural basis of the human high molecular weight-melanoma-associated antigen (HMW-MAA) mimicry by the mouse anti-idiotypic (anti-id) monoclonal antibody (mAb) MK2-23. Using x-ray crystallography, we have characterized the three-dimensional structure of the anti-id mAb MK2-23 Fab' and shown that its heavy chain complementarity-determining region (CDR3) (H3) and its light chain CDR1 (L1) are closely associated. These moieties are the source of HMW-MAA mimicry, since they display partial amino acid sequence homology along with a similar structural fold with the HMW-MAA core protein. Furthermore, a 15-residue peptide comprising the H3 loop of anti-id mAb MK2-23 demonstrates HMW-MAA-like in vitro and in vivo reactivity. This peptide in conjunction with the structural data will facilitate the characterization of the effect of the degree of antigen mimicry on the induction of a self-antigen-specific immune response by a mimic.

Estrone sulfatase (ES; 562 amino acids), one of the key enzymes responsible for maintaining high levels of estrogens in breast tumor cells, is associated with the membrane of the endoplasmic reticulum (ER). The structure of ES, purified from the microsomal fraction of human placentas, has been determined at 2.60-A resolution by x-ray crystallography. This structure shows a domain consisting of two antiparallel alpha-helices that protrude from the roughly spherical molecule, thereby giving the molecule a "mushroom-like" shape. These highly hydrophobic helices, each about 40 A long, are capable of traversing the membrane, thus presumably anchoring the functional domain on the membrane surface facing the ER lumen. The location of the transmembrane domain is such that the opening to the active site, buried deep in a cavity of the "gill" of the "mushroom," rests near the membrane surface, thereby suggesting a role of the lipid bilayer in catalysis. This simple architecture could be a prototype utilized by the ER membrane in dictating the form and the function of ER-resident enzymes.