Integrins are a family of transmembrane glycoprotein signaling receptors that can transmit bioinformation bidirectionally across the plasma membrane. Integrin αIIbβ3 is expressed at a high level in platelets and their progenitors, where it plays a central role in platelet functions, hemostasis, and arterial thrombosis. Integrin αIIbβ3 also participates in cancer progression, such as tumor cell proliferation and metastasis. In resting platelets, integrin αIIbβ3 adopts an inactive conformation. Upon agonist stimulation, the transduction of inside-out signals leads integrin αIIbβ3 to switch from a low-to high-affinity state for fibrinogen and other ligands. Ligand binding causes integrin clustering and subsequently promotes outside-in signaling, which initiates and amplifies a range of cellular events to drive essential platelet functions such as spreading, aggregation, clot retraction, and thrombus consolidation. Regulation of the bidirectional signaling of integrin αIIbβ3 requires the involvement of numerous interacting proteins, which associate with the cytoplasmic tails of αIIbβ3 in particular. Integrin αIIbβ3 and its signaling pathways are considered promising targets for antithrombotic therapy. This review describes the bidirectional signal transduction of integrin αIIbβ3 in platelets, as well as the proteins responsible for its regulation and therapeutic agents that target integrin αIIbβ3 and its signaling pathways.

The functions of heparan sulfate (HS) depend on the expression of structural domains that interact with protein partners. Glycosaminoglycans (GAGs) exhibit a high degree of polydispersity in their composition, chain length, sulfation, acetylation, and epimerization patterns. It is essential for the understanding of GAG biochemistry to produce detailed structural information as a function of spatial and temporal factors in biological systems. Toward this end, we developed a set of procedures to extract GAGs from various rat organ tissues and examined and compared HS expression levels using liquid chromatography/ mass spectrometry. Here we demonstrate detailed variations in HS GAG chains as a function of organ location. These studies shed new light on the structural variation of GAG chains with respect to average length, disaccharide composition, and expression of low abundance structural epitopes, including unsubstituted amino groups and lyase-resistant oligosaccharides. The data show the presence of a disaccharide with an unsubstituted amino group that is endogenous and widely expressed in mammalian organ tissues.

A key challenge to investigations into the functional roles of glycosaminoglycans (GAGs) in biological systems is the difficulty in achieving sensitive, stable and reproducible mass spectrometric analysis. GAGs are linear carbohydrates with domains that vary in the extent of sulfation, acetylation and uronic acid epimerization. It is of particular importance to determine spatial and temporal variations of GAG domain structures in biological tissues. In order to analyze GAGs from tissue, it is useful to couple mass spectrometry with an on-line separation system. The purposes of the separation system are both to remove components that inhibit GAG ionization and to enable the analysis of very complex mixtures. This contribution presents amide-silica hydrophilic interaction chromatography (HILIC) in a chip-based format for LC/MS of heparin, heparan sulfate and chondroitin/dermatan sulfate GAGs. The chip interface yields robust performance in the negative ion mode that is essential for GAGs and other acidic glycan classes while the built-in trapping cartridge reduces background from the biological tissue matrix. The HILIC chromatographic separation is based on a combination of the glycan chain lengths and the numbers of hydrophobic acetate groups and acidic sulfate groups. In summary, chip based amide-HILIC LC/MS is an enabling technology for GAG glycomics profiling.

Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria. However, the components that transport plant-synthesized lipids to the symbiosome membrane remain unknown. This study identified and functionally characterized the Chinese milk vetch (Astragalus sinicus) lipid transfer protein AsE246, which is specifically expressed in nodules. It was found that AsE246 can bind lipids in vitro. More importantly, AsE246 can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol in vivo. Immunofluorescence and immunoelectron microscopy showed that AsE246 and digalactosyldiacylglycerol localize in the symbiosome membrane and are present in infection threads. Overexpression of AsE246 resulted in increased nodule numbers; knockdown of AsE246 resulted in reduced nodule numbers, decreased lipids contents in nodules, diminished nitrogen fixation activity, and abnormal development of symbiosomes. AsE246 knockdown also resulted in fewer infection threads, nodule primordia, and nodules, while AsE246 overexpression resulted in more infection threads and nodule primordia, suggesting that AsE246 affects nodule organogenesis associated with infection thread formation. Taken together, these results indicate that AsE246 contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis.

Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods.