What Happens If a Human Galectin Enters the Endoplasmic Reticulum?

Mammalian galectins have no signal peptide, and it is not known what would happen if a galectin is directed to take the classical export route. The corresponding engineering of galectin-specific cDNA will answer questions on the fate of a signal peptide-bearing protein variant after its entry into the endoplasmic reticulum (ER). Affinity chromatography and mass-spectrometric analysis of occupancy of potential N-glycosylation sites for the galectin, binding and functional assays with cells as well as subcellular fractionation by density gradient ultracentrifugation and immunocytochemical colocalization with ER/Golgi markers report on aspects of the consequences of letting a galectin enter new territory.

Characterizing ligand-induced conformational changes in clinically relevant galectin-1 by H/HO (DO) exchange.

Glycans of cellular glycoconjugates serve as biochemical signals for a multitude of (patho)physiological processes via binding to their receptors (e.g. lectins).

How presence of a signal peptide affects human galectins-1 and -4: Clues to explain common absence of a leader sequence among adhesion/growth-regulatory galectins.

Background: Galectins are multifunctional effectors, which all share absence of a signal sequence. It is not clear why galectins belong to the small set of proteins, which avoid the classical export route.

Methods: Products of recombinant galectin expression in P.

Design-functionality relationships for adhesion/growth-regulatory galectins.

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface.

Chicken GRIFIN: Structural characterization in crystals and in solution.

Despite its natural abundance in lenses of vertebrates the physiological function(s) of the galectin-related inter-fiber protein (GRIFIN) is (are) still unclear. The same holds true for the significance of the unique interspecies (fish/birds vs mammals) variability in the capacity to bind lactose. In solution, ultracentrifugation and small angle X-ray scattering (at concentrations up to 9 mg/mL) characterize the protein as compact and stable homodimer without evidence for aggregation.

Application of Hydrogen/Deuterium Exchange-Mass Spectrometry to Biopharmaceutical Development Requirements: Improved Sensitivity to Detection of Conformational Changes.

The usefulness of the higher-order structure information provided by hydrogen/deuterium exchange mass spectrometry (HDX-MS) in the protein therapeutic field is undisputed; however, its applicability as a method for critical quality and comparability assessment has until now not been demonstrated. Here we present results demonstrating for the first time the applicability of the HDX-MS technique to monitor structural changes due to methionine oxidation at sensitivity levels realistic to the requirements of biopharmaceutical research and development. For the analyzed heavy chain marker peptides deuterium uptake differences due to oxidation at the conserved methionine in position 254 were significantly verifiable at the lowest increase (1%) through spiked oxidized IgG1.

Mapping the Interactions of Selective Biochemical Probes of Antibody Conformation by Hydrogen-Deuterium Exchange Mass Spectrometry.

Protein-based pharmaceuticals represent the fastest growing group of drugs in development in the pharmaceutical industry. One of the major challenges in the discovery, development, and distribution of biopharmaceuticals is the assessment of changes in their higher-order structure due to chemical modification. Here, we investigated the interactions of three different biochemical probes (F s) generated to detect conformational changes in a therapeutic IgG1 antibody (mAbX) by local hydrogen-deuterium exchange mass spectrometry (HDX-MS).

Development, preclinical safety, formulation, and stability of clinical grade bevacizumab-800CW, a new near infrared fluorescent imaging agent for first in human use.

There is a dire need for better visualization of cancer and analysis of specific targets in vivo. Molecular imaging with fluorescence is gaining more and more attention, as it allows detection of these targets and has advantages over radioactivity, such as no radiation dose, and lower costs. A key challenge in optical imaging however, is translation of the newly developed tracers from pre-clinical phase to clinical application.

Combining Crystallography and Hydrogen-Deuterium Exchange to Study Galectin-Ligand Complexes.

The physiological significance arising from translating information stored in glycans into cellular effects explains the interest in structurally defining lectin-carbohydrate recognition. The relatively small set of adhesion/growth-regulatory galectins in chicken makes this system attractive to study the origins of specificity and divergence. Cell binding by using glycosylation mutants reveals binding of the N-terminal domain of chicken galectin-8 (CG-8N) to α-2,3-sialylated and galactose-terminated glycan chains.

New open-chain tetrapyrroles as chromophores in the plant photoreceptor phytochrome.

A series of six open-chain tetrapyrroles has been synthesized and used as chromophores for the plant photoreceptor protein phytochrome. The novel chromophores vary in the size of substituents 17 and 18 at ring D. This ring undergoes maximal conformational change upon light excitation ( Z --> E photoisomerization of the 15,16-double bond).

Femtosecond kinetics of photoconversion of the higher plant photoreceptor phytochrome carrying native and modified chromophores.

The photoprocesses of native (phyA of oat), and of C-terminally truncated recombinant phytochromes, assembled instead of the native phytochromobilin with phycocyanobilin (PCB-65 kDa-phy) and iso-phycocyanobilin (iso-PCB-65 kDa-phy) chromophores, have been studied by femtosecond transient absorption spectroscopy in both their red absorbing phytochrome (P(r)) and far-red absorbing phytochrome (P(fr)) forms. Native P(r) phytochrome shows an excitation wavelength dependence of the kinetics with three main picosecond components. The formation kinetics of the first ground-state intermediate I(700), absorbing at approximately 690 nm, is mainly described by 28 ps or 40 ps components in native and PCB phytochrome, respectively, whereas additional approximately 15 and 50 ps components describe conformational dynamics and equilibria among different local minima on the excited-state hypersurface.

Phytochromes with noncovalently bound chromophores: the ability of apophytochromes to direct tetrapyrrole photoisomerization.

Chromophore-apoprotein interactions were studied with recombinant apoproteins, oat phytochrome (phyA) and CphB of the cyanobacterium Calothrix PCC7601, which were both incubated with the bilin compounds biliverdin (BV) IXalpha, phycocyanobilin (PCB) and the 3'-methoxy derivative of PCB. Previously it was shown that CphB and its homolog in Calothrix, CphA, show strong sequence similarities with each other and with the phytochromes of higher and lower plants, despite the fact that CphB carries a leucine instead of a cysteine at the chromophore attachment position and thus holds the chromophore only noncovalently. CphA binds tetrapyrrole chromophores in a covalent, phytochrome-like manner.