Under homeostatic conditions, animals use well-defined hypothalamic neural circuits to help maintain stable body weight, by integrating metabolic and hormonal signals from the periphery to balance food consumption and energy expenditure. In stressed or disease conditions, however, animals use alternative neuronal pathways to adapt to the metabolic challenges of altered energy demand. Recent studies have identified brain areas outside the hypothalamus that are activated under these 'non-homeostatic' conditions, but the molecular nature of the peripheral signals and brain-localized receptors that activate these circuits remains elusive. Here we identify glial cell-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) as a brainstem-restricted receptor for growth and differentiation factor 15 (GDF15). GDF15 regulates food intake, energy expenditure and body weight in response to metabolic and toxin-induced stresses; we show that Gfral knockout mice are hyperphagic under stressed conditions and are resistant to chemotherapy-induced anorexia and body weight loss. GDF15 activates GFRAL-expressing neurons localized exclusively in the area postrema and nucleus tractus solitarius of the mouse brainstem. It then triggers the activation of neurons localized within the parabrachial nucleus and central amygdala, which constitute part of the 'emergency circuit' that shapes feeding responses to stressful conditions. GDF15 levels increase in response to tissue stress and injury, and elevated levels are associated with body weight loss in numerous chronic human diseases. By isolating GFRAL as the receptor for GDF15-induced anorexia and weight loss, we identify a mechanistic basis for the non-homeostatic regulation of neural circuitry by a peripheral signal associated with tissue damage and stress. These findings provide opportunities to develop therapeutic agents for the treatment of disorders with altered energy demand.

Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase‐catalyzed glycosylation of the best‐selling biotherapeutic Herceptin, an anti‐HER2 antibody. Precise MS analysis of the intact four‐chain Ab heteromultimer reveals nonspecific, non‐enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non‐natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or “glycorandomization”) were readily generated.

The reaction of 1,1-diphenylhydrazine with Ti(NMe2)2Cl2 produced the monomeric terminal titanium hydrazido(2-) species Ti(NNPh2)Cl2(HNMe2)2 (1) in near-quantitative yield. The reaction of Ti(NMe2)2Cl2 with the less sterically demanding ligand precursors 1,1-dimethylhydrazine or N-aminopiperidine gave the dimeric mu-eta2,eta1-bridged compounds Ti2(mu-eta2,eta1-NNMe2)2Cl4(HNMe2)2 (2) and Ti2[mu-eta2,eta1-NN(CH2)5]2Cl4(HNMe2)3 (3). The X-ray structures of 2 and 3 showed the formation of N-H...Cl hydrogen bonded dimers or chains, respectively. The reaction of 1 with an excess of pyridine formed [Ti(NNPh2)Cl2(py)2]n (4, n = 1 or 2). The reaction of the tert-butyl imido complex Ti(N(t)Bu)Cl2(py)3 with either 1,1-dimethylhydrazine or N-aminopiperidine again resulted in the formation of hydrazido-bridged dimeric complexes, namely Ti2(mu-eta2,eta1-NNMe2)2Cl4(py)2 (5, structurally characterized) and Ti2[mu-eta2,eta1-NN(CH2)5]2Cl4(py)2 (6). Compounds 1 and 4 are potential new entry points into terminal hydrazido(2-) chemistry of titanium. Compound 1 reacted with neutral fac-N3 donor ligands to form Ti(NNPh2)Cl2(Me3[9]aneN3) (7), Ti(NNPh2)Cl2(Me3[6]aneN3) (8), Ti(NNPh2)Cl2[HC(Me2pz)3] (9, structurally characterized), and Ti(NNPh2)Cl2[HC(n)Bupz)3] (10) in good yields (Me3[9]aneN3 = trimethyl-1,4,7-triazacyclononane, Me3[6]aneN3 = trimethyl-1,3,5-triazacyclohexane, HC(Me2pz)3 = tris(3,5-dimethylpyrazolyl)methane, and HC((n)Bupz)3 = tris(4-(n)butylpyrazolyl)methane). DFT calculations were performed on both the model terminal hydrazido compound Ti(NNPh2)Cl2[HC(pz)3] (I) and the corresponding imido compounds Ti(NMe)Cl2[HC(pz)3] (II) and Ti(NPh)Cl2[HC(pz)3] (III). The NNPh2 ligand binds to the metal center in an analogous manner to that of terminal imido ligands (metalligand triple bond), but with one of the Ti=N(alpha) pi components significantly destabilized by a pi interaction with the lone pair of the N(beta) atom. The NR ligand sigma donor ability was found to be NMe > NPh > NNPh2, whereas the overall (sigma + pi) donor ability is NMe > NNPh2 > NPh, as judged by fragment orbital populations, Ti-N atom-atom overlap populations, and fragment-charge analysis. DFT calculations on the hydrazido ligand in a mu-eta2,eta1-bridging mode showed involvement of the N=N pi electrons in donation to one of the Ti centers. This TiN2 interaction is best represented as a metallocycle.

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man 8 -9 Xyl 1-2 Me 3 GlcNAc 2 . The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins.

The complete degradation of N-linked glycans by the pathogenic bacterium Streptococcus pneumoniae is facilitated by the large multimodular cell wall-attached exo-β-D-N-acetylglucosaminidase StrH. Structural dissection of this virulence factor using X-ray crystallography showed it to have two structurally related glycoside hydrolase family 20 catalytic domains, which displayed the expected specificity for complex N-glycans terminating in N-acetylglucosamine but exhibited unexpected differences in their preferences for the substructures present in these glycans. The structures of the two catalytic domains in complex with unhydrolyzed substrates, including an N-glycan possessing a bisecting N-acetylglucosamine residue, revealed the specific architectural features in the active sites that confer their differential specificities. Inhibitors of StrH are demonstrated to be effective tools in modulating the interaction of StrH with components of the host, such as the innate immune system. Overall, new structural and functional insight into a carbohydrate-mediated component of the pneumococcus-host interaction is provided.