The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.

Accumulation of degenerated elastic fibers in the sun-exposed skin designated as actinic elastosis is a histological hallmark of photodamaged skin. Previous studies have indicated that the elastic fibers of actinic elastosis interact with lysozyme and are modified by N(ɛ)-(carboxymethyl)lysine (CML), one of the major advanced glycation end products (AGEs). We studied here how CML modification of elastin is involved in the pathogenesis of actinic elastosis. The CML-modified insoluble elastin became resistant to neutrophil elastase digestion, which was reversed by treatment with aminoguanidine, a potent inhibitor of AGE formation. In a temperature-dependent aggregation assay, CML-modified elastin rapidly formed self-aggregates, the size of which was larger than unmodified elastin. The elastic fiber sheets prepared from CML-modified α-elastin showed 3D wider diameter, tortuous appearance, and decreased elasticity on tensile tests. The CML-modified α-elastin, but not unmodified α-elastin, was found to bind to lysozyme in vitro, supporting the immunohistochemical findings that the antibodies for lysozyme and CML reacted simultaneously with the elastic fibers of actinic elastosis and UV-irradiated skin. The glycated elastin is likely to cause the accumulation of abnormally aggregated elastic fibers and unusual interaction with lysozyme in actinic elastosis.

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CD22 (Siglec-2) is a B-cell membrane-bound lectin that recognizes glycan ligands containing a2,6-linked sialic acid (a2,6Sia) and negatively regulates signaling through the B-cell Ag receptor (BCR). Although CD22 has been investigated extensively, its precise function remains unclear due to acting multiple phases. Here, we demonstrate that CD22 is efficiently activated in trans by complexes of Ag and soluble IgM (sIgM) due to the presence of glycan ligands on sIgM. This result strongly suggests sIgM as a natural trans ligand for CD22. Also, CD22 appears to serve as a receptor for sIgM, which induces a negative feedback loop for B-cell activation similar to the Fc receptor for IgG (FccRIIB).Key words: B cells . CD22 . Fc receptor . IgM . Siglec-2 Supporting Information available online Introduction CD22 is a 140 kDa glycoprotein on the surface of B cells that negatively regulates signaling through the B-cell Ag receptor (BCR) [1][2][3]. There are six tyrosine residues within the cytoplasmic portion of CD22, four of which are located within ITIMs [4]. These tyrosine residues are phosphorylated upon BCR cross-linking, leading to recruitment of SHP-1 [4,5]. SHP-1 subsequently dephosphorylates the BCR-proximal signaling molecules, resulting in downmodulation of BCR signaling. Consistent with this, B cells from CD22-deficient mice are hyperactive [6][7][8][9]. The extracellular portion of CD22 is composed of seven immunoglobulin (Ig)-like domains, the most distal of which is a V-set Ig-like domain that recognizes a2,6-linked sialic acid (a2,6Sia)-containing glycoconjugates [3,10]. a2,6Sia is common at the terminal of N-linked glycans and is abundantly expressed on various kinds of cells, including erythrocytes, monocytes, B cells, and T cells. a2,6Sia also exists on soluble plasma proteins such as serum-soluble IgM (sIgM) [11]. CD22 is a member of the sialic acid-binding Ig-like lectin (Siglec) family, Results and discussion sIgM binds to CD22 on the cells in a b-galactoside a2,6-sialyltransferase I-dependent manner Since sIgM has been shown to bind to recombinant CD22 fusion protein [11], we tested whether sIgM binds to CD22-expressing cells. The mouse myeloma line J558L fails to express the CD22 glycan ligand a2,6Sia at the terminal of N-glycan due to a lack of b-galactoside a2,6-sialyltransferase I (ST6GalI) expression. Introduction of a ST6GalI expression vector can restore a2,6Sia on cell-surface glycoproteins and we showed previously that the soluble CD22 fusion protein (CD22-Fc) bound to J558L cells expressing ST6GalI (J558L/ST6) but not to J558L cells [16]. Since sIgM has been identified as a potential glycan ligand for CD22 [11], we examined whether sIgM binds to these cells. (4-Hydroxy-3-nitrophenyl) acetyl (NP)-specific sIgM bound to Ag NP-PE (Ag/sIgM) was able to bind to CD22-expresssing (J558L/CD22) cells but not to CD22-deficient (J558L) cells (Fig. 1A). Double staining with anti-CD22 mAb is shown in Supporting Information Fig. 1. This binding was not prevented by the presence of FCS containing a2,6Sia,...

The precursor protein of localized cutaneous amyloidosis (LCA) is believed to be cytokeratins on the basis of previous immunohistochemical studies. To identify the candidate amyloid protein biochemically, amyloid proteins were extracted with distilled water from lesional skin of LCA associated with Bowen's disease. The proteins were resolved on one- or two-dimensional polyacrylamide gel electrophoresis followed by characterization with immunoblot analysis. The proteins with multiple molecular weights of 50-67 kDa and two proteins with 25 and 35 kDa were identified as keratins, serum amyloid P component and apolipoprotein E, respectively. The unknown 14-kDa (pI = 7.0) and 42-kDa (pI = 5.4) proteins reacted with the antibody against galectin-7 and actin, respectively. The protein with the molecular weight of 14 kDa was identified as galectin-7 by MALDI-TOF mass spectrometer. Their electrophoretic mobilities were identical with normal counterparts extracted from cultured normal human keratinocytes. Galectin-7 and actin were detected by immunoblot assay in the water-soluble fractions prepared from the lesional skins of two patients with primary LCA. Immunohistochemical studies of tumor-associated (n = 9) and primary (n = 10) LCA revealed various degrees of positive immunoreactivities with the antibodies for galectin-7 and F-actin. Galectin-7 and actin, which contain considerable amount of β-sheet structure, may be candidate amyloidogenic proteins of primary and secondary LCA.