A Human Lectin Microarray for Sperm Surface Glycosylation Analysis

Abstract

Glycosylation is one of the most abundant and functionally important protein post-translational modifications. As such, technology for efficient glycosylation analysis is in high demand. Lectin microarrays are a powerful tool for such investigations and have been successfully applied for a variety of glycobiological studies. However, most of the current lectin microarrays are primarily constructed from plant lectins, which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans. Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane-associated proteins including heat shock protein 90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays. Molecular & Cellular

Human Erythrocyte Surface Fucose Expression Increases with Age and Hyperglycemia

“…Washed RBC were subjected to ice cold hypotonic lysis in 20 mM Tris, pH 7.6 and protease inhibitor (05056489001, Roche) (31). Lysates were washed three times in hypotonic lysis buffer (37000 g, 4°C, 30 minutes) before resuspension in minimal hypotonic lysis buffer.…”

“…Ghost preparations were mixed in equal volumes with SDS sample buffer containing 8M urea (31) and heated at 100°C for 10 minutes. Ghost protein samples were fractionated by gel electrophoresis using…”

“…N-and O-glycome pro les of cell surfaces can be full analysed my mass spectrometry (28) as well as state of art technology such as solid phase chemoenzymatic method (29). Human and plant lectins are being becoming increasingly popular for studies of cell surface changes, as they offer direct binding to speci c terminal glycans (30,31). The use of lectins with ow cytometry to determine cell surface glycosylation expression has the advantage of accessing all cell surface glycans available for binding, thus mimicking cell-cell interactions, contrasting with conventional techniques such as PAS staining.…”

Integrated Strategies to Gain a Systems-Level View of Dynamic Signaling Networks

“…Huang et al, 2004; Kung et al, 2009). Importantly, because they are also amenable to activity assays designed to determine enzyme-substrate relationships, functional protein microarrays have been particularly useful for determining the global substrate specificity of key signaling enzymes(Cox et al, 2015; Jeong, Rho, & Zhu, 2011; Lu, Lin, Boeke, & Zhu, 2013; Sun et al, 2016). For example, to gain a better understanding of the organization of human phosphorylation networks, we recently examined the global substrate profiles of 289 unique human kinases using functional protein microarrays composed of ~4,200 full-length human proteins (Newman et al, 2013).…”

“…This information will be particularly useful for understanding the functional consequences of phosphorylation and for developing new tools, such as phospho-specific antibodies and novel FRET-based kinase activity reporters, used to study dynamic phosphorylation networks. Moreover, the generalizability of this approach suggests that it will be useful for examining the global substrate profiles of enzymes involved in SUMOylation (Cox et al, 2015), ubiquitylation (Jeong et al, 2011), acetylation (Lu et al, 2013) and O-glycosylation (Sun et al, 2016), which together will offer clues about potential points of intersection between different signaling modalities.…”