TGFß counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides.

During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4-25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFß in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes. KEY MESSAGES : sHA promotes lymphangiogenesis primarily through increased LEC proliferation sHA induces proliferation in a narrow concentration window due to upregulated TGFß Smaller HA oligosaccharides more potently induce proliferation than larger ones VEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHA Sialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC.

Genetic code expansion for multiprotein complex engineering.

We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies.

Chemical Synthesis of Glycosaminoglycans.

Glycosaminoglycans (GAGs) as one major part of the glycocalyx are involved in many essential biological cell processes, as well as in many courses of diseases. Because of the potential therapeutic application of GAG polymers, fragments, and also derivatives toward different diseases (e.g., heparin derivatives against Alzheimer's disease), there is a continual growing demand for new chemical syntheses, which suffice the high claim to stereoselectivity and chemoselectivity. This Review summarizes the progress of chemical syntheses of GAGs over the last 10 years. For each class of the glycosaminoglycans-hyaluronan (HA), heparan sulfate/heparin (HS/HP), chondroitin/dermatan sulfate (CS/DS), and keratan sulfate (KS)-mainly novel glycosylation strategies, elongation sequences, and protecting group patterns are discussed, but also (semi)automated syntheses, enzymatic approaches, and functionalizations of synthesized or isolated GAGs are considered.

Bioconjugation via azide-Staudinger ligation: an overview.

Bioconjugation techniques using organic azides are compared in this critical review. A particular focus is on chemical ligation reactions and their application to chemical biology (179 references).

Synthesis-guided structure revision of the sarcodonin, sarcoviolin, and hydnellin natural product family.

A sweeping structural revision of the sarcodonin natural product family (published structures 1a-13a) is proposed after extensive studies aimed at their chemical synthesis. Key features of revised structure 1b include replacement of the N,N-dioxide moiety with an oxime, ring-opening of the central diketopiperazine, and transposition of the terphenyl wing from the 1ß-2ß position of 1a to the 2ß-3ß position of 1b. This structure revision arose from the serendipitous synthesis of a benzodioxane aminal (44) whose structure was unambiguously determined by X-ray crystallography and whose spectral properties bore considerable resemblance to the published data for the sarcodonins. A versatile new method for O-arylation of hydroxamic acids is also reported herein, as well as a manganese(III)-mediated α-oxidation of hydroxamic acids to aminals.

The Glc2Man2-fragment of the N-glycan precursor--a novel ligand for the glycan-binding protein malectin?

The Glcalpha(1-->3)Glcalpha(1-->3)Manalpha(1-->2)Man tetrasaccharide (Glc(2)Man(2)-fragment), a substructure of the natural N-glycan precursor, was synthesized. The interaction of this fragment with the protein malectin, a carbohydrate binding protein localized in the endoplasmatic reticulum, was investigated by (1)H(15)N HSQC experiments and isothermal calorimetry. The chemical shift perturbations of nuclei in the protein's backbone caused by the binding of the Glc(2)Man(2)-fragment to malectin suggest a binding mode like the known ligand nigerose.

New methods for the generation of carbohydrate arrays on glass slides and their evaluation.

Glycosides, having spacers functionalized with an aldehyde or a carboxylic group, were immobilized through reductive amination or amidation, respectively, onto amino-functionalized glass slides. Hybridization experiments with lectins exhibited very little nonspecific protein binding, hence precluding the necessity for the blocking of unreacted functional groups on the glass slide. The covalency and the concentration dependency of the sugar ligation to the glass slide were demonstrated; the reversibility and the selectivity of lectin-carbohydrate interactions were shown.