Galectin-1 and galectin-8 have redundant roles in promoting plasma cell formation.

Galectin (Gal) family members are a type of soluble lectin, and they play important roles in immunomodulation. Their redundant roles have been proposed. We previously found that Gal-1 promotes the formation of Ab-secreting plasma cells, but B cells from Gal-1-deficient and control animals produce comparable amounts of Abs. In the current study, we used synthetic sulfomodified N-acetyllactosamine (LacNAc) analogs and short hairpin RNAs for Gal-8 to demonstrate a redundancy in the effects of Gal-1 and Gal-8 on plasma cell formation. Gal-1 and Gal-8 were both expressed during plasma cell differentiation, and both Gals promoted the formation of plasma cells. Gal-1 and Gal-8 bound better to mature B cells than to plasma cells, and the expression of glycosyltransferase enzymes changed during differentiation, with a decrease in mannosyl (α-1,6-)-glycoprotein ß-1,6-N-acetyl-glucosaminyltransferase and N-acetylglucosaminyltransferase-1 mRNAs in plasma cells. Synthetic sulfomodified Galß1-3GlcNAc disaccharides (type 1 LacNAcs) selectively prevented Gal-8 binding, leading to a blockade of Ab production in Gal-1-deficient B cells. Furthermore, synthetic type 1 LacNAcs that were able to block the binding of both Gals greatly reduced the effect of exogenously added recombinant Gal-1 and Gal-8 on promoting Ab production. These results reveal a novel role for Gal-8 in collaboration with Gal-1 in plasma cell formation, and suggest the possibility of using distinct LacNAc ligands to modulate the function of Gals.

Modulation of substrate specificities of D-sialic acid aldolase through single mutations of Val-251.

In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RS-aldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (α/ß)(8)-barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase·l-arabinose and RS-aldolase·hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C(6) to C(10) for the design of antagonists and inhibitors of glycoenzymes.

Precise Probing of Residue Roles by Post-Translational ß,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites.

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-ß,γ C-N bond formation. Installation of dehydroalanine (as a "tag") allowed the testing of nitrogen conjugate nucleophiles in "aza-Michael"-1,4-additions (to "modify"). This allowed the creation of a regioisomer of His (iso-His, Hisiso) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-Hisiso. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from Bacillus lentus and Mycobacterium tuberculosis pantothenate synthetase.

Ion mobility-mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility.

Rotary ATPases play fundamental roles in energy conversion as their catalytic rotation is associated with interdomain fluctuations and heterogeneity of conformational states. Using ion mobility mass spectrometry we compared the conformational dynamics of the intact ATPase from Thermus thermophilus with those of its membrane and soluble subcomplexes. Our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. To provide a structural context for our experimental data we performed molecular dynamics simulations and observed conformational changes of the peripheral stalks that reflect their intrinsic flexibility. By isolating complexes at different phases of cell growth and manipulating nucleotides, metal ions and pH during isolation, we reveal differences that can be related to conformational changes in the Vo complex triggered by ATP binding. Together these results implicate nucleotides in modulating flexibility of the stator components and uncover mechanistic detail that underlies operation and regulation in the context of the holoenzyme.

Synthesis and characterization of sulfated Gal-ß-1,3/4-GlcNAc disaccharides through consecutive protection/glycosylation steps.

We have developed an expeditious procedure to yield large amounts of orthogonally protected Gal-ß1,3/4-GlcNAc, which allowed for the systematic introduction of a sulfate group onto the C3/C6 positions of Gal and/or the C6 position of GlcNAc. In particular, the disaccharide precursors were prepared in five or six steps and high overall yield from para-tolyl-6-O-tert-butyldiphenylsilyl-1-thio-ß-D-galactopyranoside. After deprotection and sulfation steps, the final products were characterized by using several NMR methods to unambiguously confirm the location of each introduced sulfate group and they were examined for their binding specificity of human galectin-1 and galectin-8.