Extracellular Glycoprotein Research Articles

Thrombospondins: Conserved mediators and modulators of metazoan extracellular matrix.

This review provides a personal overview of significant scientific developments in the thrombospondin field during the course of my career. Thrombospondins are multidomain, multimeric, calcium-binding extracellular glycoproteins with context-specific roles in tissue organisation. They act at cell surfaces and within ECM to regulate cell phenotype and signalling, differentiation and assembly of collagenous ECM, along with tissue-specific roles in cartilage, angiogenesis and synaptic function. More recently, intracellular, homeostatic roles have also been identified. Resolution of structures for the major domains of mammalian thrombospondins has facilitated major advances in understanding thrombospondin biology from molecule to tissue; for example, in illuminating molecular consequences of disease-causing coding mutations in human pseudoachrondroplasia. Although principally studied in vertebrates, thrombospondins are amongst the most ancient of animal ECM proteins, with many invertebrates encoding a single thrombospondin and the thrombospondin gene family of vertebrates originating through gene duplications. Moreover, thrombospondins form one branch of a thrombospondin superfamily that debuted at the origin of metazoans. The super-family includes additional sub-groups, present only in invertebrates, that differ in N-terminal domain organisation, share the distinctive TSP C-terminal region domain architecture and, to the limited extent studied to date, apparently contribute to tissue development and organisation. Finally, major lines of translational research are discussed, related to fibrosis; TSP1, TSP2 and inhibition of angiogenesis; and the alleviation of chronic cartilage tissue pathologies in pseudoachrondroplasia.

OsGLP8-7 interacts with OsPRX111 to detoxify excess copper in rice

Lignin is a phenolic biopolymer generated from phenylpropanoid pathway in the secondary cell wall and is required for defense of plants against various stress. Although the fact of stress-induced lignin deposition has been clearly demonstrated, it remains largely elusive how the formation of lignin is promoted under Cu stress. The present study showed that OsGLP8-7, an extracellular glycoprotein of rice (Oryza sativa L.), plays an important function against Cu stress. The loss function of OsGLP8-7 results in Cu sensitivity whereas overexpression of OsGLP8-7 scavenges Cu-induced superoxide anion (O2•ˉ). OsGLP8-7 interacts with apoplastic peroxidase111 (OsPRX111) and elevates OsPRX111 stability when exposed to excess Cu. In OsGLP8-7 overexpressing (OE) lines, the retention of Cu within cell wall limiting Cu uptake into cytoplasm is attributed to the enhanced lignification required for Cu tolerance. Exogenous application of a lignin inhibitor can impair the Cu tolerance of transgenic Arabidopsis lines overexpressing OsGLP8-7. In addition, co-expression of OsGLP8-7 and OsPRX111 genes in tobacco leaves leads to an improved lignin deposition compared to leaves expressing each gene individually or the empty vector. Taken together, our findings provided the convincing evidences that the interaction between OsGLP8-7 and OsPRX111 facilitates effectively lignin polymerization, thereby contributing to Cu tolerance in rice.

Safety of Anti-Reelin Therapeutic Approaches for Chronic Inflammatory Diseases.

Reelin, a large extracellular glycoprotein, plays critical roles in neuronal development and synaptic plasticity in the central nervous system (CNS). Recent studies have revealed non-neuronal functions of plasma Reelin in inflammation by promoting endothelial-leukocyte adhesion through its canonical pathway in endothelial cells (via ApoER2 acting on NF-κB), as well as in vascular tone regulation and thrombosis. In this study, we have investigated the safety and efficacy of selectively depleting plasma Reelin as a potential therapeutic strategy for chronic inflammatory diseases. We found that Reelin expression remains stable throughout adulthood and that peripheral anti-Reelin antibody treatment with CR-50 efficiently depletes plasma Reelin without affecting its levels or functionality within the CNS. Notably, this approach preserves essential neuronal functions and synaptic plasticity. Furthermore, in mice induced with experimental autoimmune encephalomyelitis (EAE), selective modulation of endothelial responses by anti-Reelin antibodies reduces pathological leukocyte infiltration without completely abolishing diapedesis. Finally, long-term Reelin depletion under metabolic stress induced by a Western diet did not negatively impact the heart, kidney, or liver, suggesting a favorable safety profile. These findings underscore the promising role of peripheral anti-Reelin therapeutic strategies for autoimmune diseases and conditions where endothelial function is compromised, offering a novel approach that may avoid the immunosuppressive side effects associated with conventional anti-inflammatory therapies.

Sialic acid in the regulation of blood cell production, differentiation and turnover.

Sialic acid is a unique sugar moiety that resides in the distal and most accessible position of the glycans on mammalian cell surface and extracellular glycoproteins and glycolipids. The potential for sialic acid to obscure underlying structures has long been postulated, but the means by which such structural changes directly affect biological processes continues to be elucidated. Here, we appraise the growing body of literature detailing the importance of sialic acid for the generation, differentiation, function and death of haematopoietic cells. We conclude that sialylation is a critical post-translational modification utilized in haematopoiesis to meet the dynamic needs of the organism by enforcing rapid changes in availability of lineage-specific cell types. Though long thought to be generated only cell-autonomously within the intracellular ER-Golgi secretory apparatus, emerging data also demonstrate previously unexpected diversity in the mechanisms of sialylation. Emphasis is afforded to the mechanism of extrinsic sialylation, whereby extracellular enzymes remodel cell surface and extracellular glycans, supported by charged sugar donor molecules from activated platelets.

Caudally pronounced deficiencies in preplate splitting and migration underly a rostro-caudal progression of cortical lamination defects in the reeler brain.

In mammalian neocortex development, every cohort of newborn neurons is guided toward the marginal zone, leading to an "inside-out" organization of the 6 neocortical layers. This migratory pattern is regulated by the extracellular glycoprotein Reelin. The reeler mouse shows a homozygous mutation of the reelin gene. Using RNA in situ hybridization we could demonstrate that the Reelin-deficient mouse cortex (male and female) displays an increasing lamination defect along the rostro-caudal axis that is characterized by strong cellular intermingling, but roughly reproduces the "inside-out" pattern in rostral cortex, while caudal cortex shows a relative inversion of neuronal positioning ("outside-in"). We found that in development of the reeler cortex, preplate-splitting is also defective with an increasing severity along the rostro-caudal axis. This leads to a misplacement of subplate neurons that are crucial for a switch in migration mode within the cortical plate. Using Flash Tag labeling and nucleoside analog pulse-chasing, we found an according migration defect within the cortical plate, again with a progressive severity along the rostro-caudal axis. Thus, loss of one key player in neocortical development leads to highly area-specific (caudally pronounced) developmental deficiencies that result in multiple roughly opposite rostral versus caudal adult neocortical phenotypes.

Nutrient availability and stoichiometry mediate microbial effects on soil carbon sequestration in tropical forests

The persistence of soil organic carbon (SOC) is primarily driven by microbial metabolic activities; however, how microbial effects on SOC sequestration are affected by soil nutrient status remains unclear. Here, we conducted a one-year-long in situ soil incubation experiment using mesh bags (with a mesh size of 38 μm, allowing bacterial colonization and fungal hyphal penetration while preventing root penetration). This experiment involved incubating fertile sugarcane soil and infertile sand across an elevational gradient, characterized by diverse climatic and biotic conditions within a tropical forest. Biomarkers, such as phospholipid fatty acids, carbon-, nitrogen-, and phosphorus-acquiring hydrolases, glomalin-related proteins, and amino sugars, were measured to characterize the production and accumulation of microbial biomass, exo-enzymes, extracellular glycoproteins, and microbial necromass. These measurements aimed to elucidate their respective contribution to the sequestration of SOC. We found that Gram-negative bacteria dominated the microbial community composition in fertile soil, and the higher nutrient availability was related to the production and accumulation of microbial necromass via promoting microbial biomass turnover, thus enhancing the accumulation of SOC in fertile soil. This process was negatively associated with phosphorus availability and carbon- and phosphorus-acquiring enzyme activities in fertile soil. In contrast, the SOC accumulation was positively correlated with nitrogen availability and stoichiometry (including C:N and C:P), as well as moisture content in infertile sand. However, more resources were preferentially allocated to stress-tolerant fungi and Gram-positive bacteria under nutrient deficiency in infertile sand used for microbial biomass maintenance, nutrient acquisition, and environmental adaption which further aggravated the consumption of SOC, resulting in SOC loss after one year of field incubation. Our results suggest that microbial effects on SOC persistence are highly context-dependent and nutrient availability-induced changes in microbial communities and microbial resource-allocation strategies are key processes for understanding and predicting the fate of carbon in tropical forest soils.

Is tear proteome profile a predictor of developing uveitis in ANA-positive patients with oligoarticular juvenile idiopathic arthritis?

Although less than one-third of anti-nuclear antibody (ANA) positive patients with oJIA develop uveitis, ANA positivity is still the most well-known marker for assessing the risk of uveitis in oligoarticular JIA (oJIA). Therefore, novel biomarkers are needed to better assess the risk of developing uveitis. For this purpose, we performed a comparative tear proteome analysis of uveitis patients to reveal the identity of differentially regulated proteins. Tear samples were collected using the Schirmer strips in 7 oJIA and 7 oJIA patients with uveitis (oJIA-U). All oJIA-U patients had developed bilateral anterior uveitis and were inactive and topical treatment-free. The nHPLC LC-MS/MS system was used for protein identification and label-free proteome comparisons. The PANTHER and STRING analyses were carried out using UniProt accession numbers of the identified proteins. Patient characteristics, e.g., age, gender, disease duration, and treatments were similar. For protein identification, three different databases were searched. Twenty-two, 147, and 258 database searches, respectively. Of these, 15 were common to all three proteome databases. Of these 15 proteins, 10 proteins were upregulated, and 2 were downregulated, based on the twofold regulation criteria. The upregulated proteins were, namely, cystatin-S, secretoglobin family 1D member, opiorphin prepropeptide, mammaglobin-B, lysozyme C, mesothelin, immunoglobulin kappa constant, extracellular glycoprotein lacritin, beta-2-microglobulin, and immunoglobulin J chain. The downregulated proteins were dermcidin and prolactin-inducible protein. Among the differentially regulated proteins, cystatin-S was the most regulated protein with an 18-fold upregulation ratio in tear samples from uveitis patients. Here, the identities and regulation ratios of several proteins were revealed when tear samples from uveitis patients were compared to patients without uveitis. These proteins are putative biomarkers for assessing uveitis risk and require further attention.

A heterozygous ZP2 mutation causes zona pellucida defects and female infertility in mouse and human

The zona pellucida (ZP) is an extracellular glycoprotein matrix surrounding mammalian oocytes. Recently, numerous mutations in genes encoding ZP proteins have been shown to be possibly related to oocyte abnormality and female infertility; few reports have confirmed the functions of these mutations in living animal models. Here, we identified a novel heterozygous missense mutation (NM_001376231.1:c.1616C>T, p.Thr539Met) in ZP2 from a primary infertile female. We showed that the mutation reduced ZP2 expression and impeded ZP2 secretion in cell lines. Furthermore, we constructed the mouse model with the mutation (Zp2T541M) using CRISPR-Cas9. Zp2WT/T541M female mice had normal fertility though generated oocytes with the thin ZP, whereas Zp2T541M female mice were completely infertile due to degeneration of oocytes without ZP. Additionally, ZP deletion impaired folliculogenesis and caused female infertility in Zp2T541M mice. Our study not only expands the spectrum of ZP2 mutation sites but also, more importantly, increases the understanding of pathogenic mechanisms of ZP2 mutations.

The interplay between adsorption and aggregation of von Willebrand factor chains in shear flows

Von Willebrand factor (VWF) is a giant extracellular glycoprotein that carries out a key adhesive function during primary hemostasis. Upon vascular injury and triggered by the shear of flowing blood, VWF establishes specific interactions with several molecular partners in order to anchor platelets to collagen on the exposed subendothelial surface. VWF also interacts with itself to form aggregates that, adsorbed on the surface, provide more anchor sites for the platelets. However, the interplay between elongation and subsequent exposure of cryptic binding sites, self-association, and adsorption on the surface remained unclear for VWF. In particular, the role of shear flow in these three processes is not well understood. In this study, we address these questions by using Brownian dynamics simulations at a coarse-grained level of resolution. We considered a system consisting of multiple VWF-like self-interacting chains that also interact with a surface under a shear flow. By a systematic analysis, we reveal that chain-chain and chain-surface interactions coexist nontrivially to modulate the spontaneous adsorption of VWF and the posterior immobilization of secondary tethered chains. Accordingly, these interactions tune VWF’s extension and its propensity to form shear-assisted functional adsorbed aggregates. Our data highlight the collective behavior VWF self-interacting chains have when bound to the surface, distinct from that of isolated or flowing chains. Furthermore, we show that the extension and the exposure to solvent have a similar dependence on shear flow, at a VWF-monomer level of resolution. Overall, our results highlight the complex interplay that exists between adsorption, cohesion, and shear forces and their relevance for the adhesive hemostatic function of VWF.

The advance of CCN3 in fibrosis.

The extracellular matrix (ECM) is comprised of various extracellular macromolecules, including collagen, enzymes, and glycoproteins, which offer structural and biochemical support to neighboring cells. After tissue injury, extracellular matrix proteins deposit in the damaged tissue to promote tissue healing. However, an imbalance between ECM production and degradation can result in excessive deposition, leading to fibrosis and subsequent organ dysfunction. Acting as a regulatory protein within the extracellular matrix, CCN3 plays a crucial role in numerous biological processes, such as cell proliferation, angiogenesis, tumorigenesis, and wound healing. Many studies have demonstrated that CCN3 can reduce the production of ECM in tissues through diverse mechanisms thereby exerting an inhibitory effect on fibrosis. Consequently, CCN3 emerges as a promising therapeutic target for ameliorating fibrosis.

A novel Reelin construct, R36, recovered behavioral deficits in the heterozygous reeler mouse.

Reelin, a large extracellular glycoprotein, plays a critical role in prenatal brain development and postnatally in synaptic plasticity, learning and memory. Dysregulation of Reelin signaling has been implicated in several neuropsychiatric disorders including schizophrenia, autism, depression, and Alzheimer's disease. Previous studies have demonstrated that Reelin's central fragment, R3456, binds to ApoER2, inducing ApoER2 clustering and subsequent intracellular signaling. We previously reported the development of a novel luciferase complementation assay which we used to demonstrate that R3456 can lead to ApoER2 receptor dimerization. Using this same assay, we explored various smaller fragments and combinations from R3456, and we identified a construct of repeats 3 and 6 (R36) which could still elicit equivalent receptor dimerization. The purpose of this study was to test R36 for biological effects in vitro and in vivo. We show that R36 was capable of initiating intracellular signaling in primary neuronal cultures. In addition, we demonstrate that a single intracerebroventricular injection of R36 protein into a model of Reelin deficiency, the heterozygous reeler mice can significantly improve cognition. These data support a role for the new construct R36 to enhance the Reelin pathway and the future possibility of exploring gene therapy approaches with R36 in diseases characterized by reduced levels of Reelin.

Abstract 3915: RNAseq analysis reveals differentiation gene drivers as potential therapeutic targets in anaplastic thyroid cancer

Abstract Papillary thyroid cancer (PTC) is a well-differentiated and highly treatable cancer with a 5-year survival rate of almost 99%. In contrast, anaplastic thyroid cancer (ATC) lacks differentiation and loses its thyroid-like functions, including lack of response to radioiodine therapy. Due to ATC’s aggressive metastatic phenotype, it is always diagnosed as stage IV with a 5 year survival rate of less than 3%. ATC lacks targetable genetic lesions making it a difficult cancer to treat, therefore RNAseq analysis was performed in an effort to investigate the differentially expressed genes between ATC to PTC. Exploration of differential gene expression between ATC and PTC was undertaken to further elucidate the differences in expression that drive the aggressive nature of ATC phenotype. RNA was isolated from human thyroid cancer cell lines T238 (ATC) and K1 (PTC) and RNA sequencing was conducted by Azenta Life Sciences. Our analysis resulted in 4,931 significant differentially expressed genes between ATC and PTC. Looking at the gene ontology analysis, pathways involving cell differentiation, proliferation, and migration revealed 90, 70, and 47 genes differentially expressed in ATC vs PTC respectively. These pathways putatively give rise to ATC’s aggressive phenotype when compared to PTC, therefore, we narrowed down the focus from 4,931 genes to 207. Through RNAseq analysis, Homeobox D4 (HOXD4) was observed to significantly increase in ATC (7.69 log2 fold change) compared to PTC. HOXD4 is a transcription factor that plays a crucial role in cell differentiation and has been associated with a worse prognosis in cancers such as ovarian serous carcinoma. As loss of differentiation is essential to ATC’s refractory nature and therapeutic resistance, targeting pathways involved in differentiation can be crucial in making ATC amenable to therapy. Other differentiation regulatory genes identified include BLK and LAMA5. BLK (3.95 log2 fold change) is a Src family tyrosine kinase proto-oncogene that is not only involved in cell proliferation but also differentiation. LAMA5 (4.76 log2 fold change) is an extracellular glycoprotein that has been linked to differentiation, migration and metastasis. Validation of the RNAseq results was completed via western blot analysis of multiple ATC and PTC cells lines. We found that HOXD4 expression was higher in ATC cell lines T238, 8505C, and SW1736 than PTC cell lines K1, TPC1, and BCPAP. Further validation was carried out by immunohistochemistry of human thyroid tissue. Preliminary IHC on ATC, PTC, and normal thyroid patient samples showed ATC has significantly increased HOXD4 expression (27.5%) compared to PTC (8.7%) and normal thyroid tissue (2.3%). Ongoing studies in the laboratory to identify novel genetic drivers of cell differentiation and proliferation so as to discover a therapeutic target to fulfill an unmet clinical need is underway. Citation Format: Kaci Kopec, Tara Jarboe, Michael Guo, Nicole R. DeSouza, Sarnath Singh, Augustine Moscatello, Humayun K. Islam, Jan Geliebter, Raj K. Tiwari. RNAseq analysis reveals differentiation gene drivers as potential therapeutic targets in anaplastic thyroid cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3915.

Normal connectivity of thalamorecipient networks in barrel equivalents of the reeler cortex.

The reeler mouse mutant has long served as a primary model to study the development of cortical layers, which is governed by the extracellular glycoprotein reelin secreted by Cajal-Retzius cells. Because layers organize local and long-range circuits for sensory processing, we investigated whether intracortical connectivity is compromised by reelin deficiency in this model. We generated a transgenic reeler mutant (we used both sexes), in which layer 4-fated spiny stellate neurons are labeled with tdTomato and applied slice electrophysiology and immunohistochemistry with synaptotagmin-2 to study the circuitry between the major thalamorecipient cell types, namely excitatory spiny stellate and inhibitory fast-spiking (putative basket) cells. In the reeler mouse, spiny stellate cells are clustered into barrel equivalents. In these clusters, we found that intrinsic physiology, connectivity, and morphology of spiny stellate and fast-spiking, putative basket cells does not significantly differ between reeler and controls. Properties of unitary connections, including connection probability, were very comparable in excitatory cell pairs and spiny stellate/fast-spiking cell pairs, suggesting an intact excitation-inhibition balance at the first stage of cortical sensory information processing. Together with previous findings, this suggests that thalamorecipient circuitry in the barrel cortex develops and functions independently of proper cortical lamination and postnatal reelin signaling.

Surface-layer protein is a public-good matrix exopolymer for microbial community organisation in environmental anammox biofilms

Extracellular polymeric substances (EPS) are core biofilm components, yet how they mediate interactions within and contribute to the structuring of biofilms is largely unknown, particularly for non-culturable microbial communities that predominate in environmental habitats. To address this knowledge gap, we explored the role of EPS in an anaerobic ammonium oxidation (anammox) biofilm. An extracellular glycoprotein, BROSI_A1236, from an anammox bacterium, formed envelopes around the anammox cells, supporting its identification as a surface (S-) layer protein. However, the S-layer protein also appeared at the edge of the biofilm, in close proximity to the polysaccharide-coated filamentous Chloroflexi bacteria but distal to the anammox bacterial cells. The Chloroflexi bacteria assembled into a cross-linked network at the edge of the granules and surrounding anammox cell clusters, with the S-layer protein occupying the space around the Chloroflexi. The anammox S-layer protein was also abundant at junctions between Chloroflexi cells. Thus, the S-layer protein is likely transported through the matrix as an EPS and also acts as an adhesive to facilitate the assembly of filamentous Chloroflexi into a three-dimensional biofilm lattice. The spatial distribution of the S-layer protein within the mixed species biofilm suggests that it is a “public-good” EPS, which facilitates the assembly of other bacteria into a framework for the benefit of the biofilm community, and enables key syntrophic relationships, including anammox.

Effect of the Reelin-Dab1 signaling pathway on the abnormal metabolism of Aβ protein induced by aluminum.

Aluminum (Al) is a common neurotoxic element that can exacerbate intracellular β-amyloid (Aβ) deposition. Reelin is a highly conserved extracellular glycoprotein that is involved in intracellular Aβ deposition. However, the action of Reelin on aluminum-induced Aβ deposition is not fully understood. Here, we investigated the effects of the Reelin-Dab1 signaling pathway on Aβ deposition in aluminum maltol (Al(mal)3) exposure in rat pheochromocytoma-derived cells (PC12). Our results showed that Al(mal)3 exposure decreased activity of PC12, increased expression of Aβ42, and decreased expression of Aβ40. Moreover, Al(mal)3 exposure in PC12 induced Reelin-Dab1 signaling pathway-associated proteins changed, decreased expression of Reelin and Dab1, and increased expression of pdab1. Moreover, the expression of Reelin, Dab1, and Aβ40 was found to be elevated in PC12 exposed to Al(mal)3 and corticosterone compared to those exposed to Al(mal)3. Also, the expression of Reelin, Dab1, and Aβ40 was found to be depressed in PC12 exposed to Al(mal)3 and streptozotocin compared with cells exposed to Al(mal)3 alone. These results suggested that Al(mal)3 inhibits the expression of the Reelin-Dab1 signaling pathway, promoting Aβ deposition. Thus, our findings provided important evidence to better understand how the Reelin-Dab1 signaling pathway may be a potential mechanism of Aβ deposition induced by aluminum.

Imaging of Indocyanine Green-Human Serum Albumin (ICG-HSA) Complex in Secreted Protein Acidic and Rich in Cysteine (SPARC)-Expressing Glioblastoma.

Glioblastoma is the most common and fatal primary glioma and has a severe prognosis. It is a challenge for neurosurgeons to remove brain tumor tissues completely by resection. Meanwhile, fluorescence-guided surgery (FGS) is a technique used in glioma surgery to enhance the visualization of tumor edges to clarify the extent of tumor resection. Indocyanine green (ICG) is the only FDA-approved NIR fluorescent agent. It non-covalently binds to human serum albumin (HSA). Secreted protein acidic and rich in cysteine (SPARC) is an extracellular glycoprotein expressed in gliomas and binds to albumin, suggesting that it plays an important role in tumor uptake of the ICG-HSA complex. Here we demonstrate the binding properties of HSA or SPARC to ICG using surface plasmon resonance and saturation binding assay. According to in vitro and in vivo studies, the results showed that the uptake of ICG-HSA complex was higher in SPARC-expressing glioblastoma cell line and tumor region compared with the uptake of free ICG. Here, we visualized the SPARC-dependent uptake of ICG and ICG-HSA complex in U87MG. Our results demonstrated that the ICG-HSA complex is likely to be used as an efficient imaging agent targeting SPARC-expressing tumors, especially glioblastoma.

Role of follistatin-like protein 1 in liver diseases.

Liver diseases, including viral hepatitis, fatty liver, metabolic-associated fatty liver disease, liver cirrhosis, alcoholic liver disease, and liver neoplasms, are major global health challenges. Despite the continued development of new drugs and technologies, the prognosis of end-stage liver diseases, including advanced liver cirrhosis and liver neoplasms, remains poor. Follistatin-like protein 1 (FSTL1), an extracellular glycoprotein, is secreted by various cell types. It is a glycoprotein that belongs to the family of secreted proteins acidic and rich in cysteine (SPARC). It is also known as transforming growth factor-beta inducible TSC-36 and follistatin-related protein (FRP). FSTL1 plays a key role in cell survival, proliferation, differentiation, and migration, as well as the regulation of inflammation and immunity. Studies have demonstrated that FSTL1 significantly affects the occurrence and development of liver diseases. This article reviews the role and mechanism of FSLT1 in liver diseases.

Characterization of the dystrophin-associated protein complex by mass spectrometry.

The dystrophin-associated protein complex (DAPC) is a highly organized multiprotein complex that plays a pivotal role in muscle fiber structure integrity and cell signaling. The complex is composed of three distinct interacting subgroups, intracellular peripheral proteins, transmembrane glycoproteins, and extracellular glycoproteins subcomplexes. Dystrophin protein nucleates the DAPC and is important for connecting the intracellular actin cytoskeletal filaments to the sarcolemma glycoprotein complex that is connected to the extracellular matrix via laminin, thus stabilizing the sarcolemma during muscle fiber contraction and relaxation. Genetic mutations that lead to lack of expression or altered expression of any of the DAPC proteins are associated with different types of muscle diseases. Hence characterization of this complex in healthy and dystrophic muscle might bring insights into its role in muscle pathogenesis. This review highlights the role of mass spectrometry in characterizing the DAPC interactome as well as post-translational glycan modifications of some of its components such as α-dystroglycan. Detection and quantification of dystrophin using targeted mass spectrometry are also discussed in the context of healthy versus dystrophic skeletal muscle.

In vivo structural modification of type II arabinogalactans with fungal endo-β-1, 6-galactanase in Arabidopsis.

Arabinogalactan-proteins (AGPs) are mysterious extracellular glycoproteins in plants. Although AGPs are highly conserved, their molecular functions remain obscure. The physiological importance of AGPs has been extensively demonstrated with β-Yariv reagent, which specifically binds to AGPs and upon introduction into cells, causes various deleterious effects including growth inhibition and programmed cell death. However, structural features of AGPs that determine their functions have not been identified with β-Yariv reagent. It is known that AGPs are decorated with large type II arabinogalactans (AGs), which are necessary for their functions. Type II AGs consist of a β-1,3-galactan main chain and β-1,6-galactan side chains with auxiliary sugar residues such as L-arabinose and 4-O-methyl-glucuronic acid. While most side chains are short, long side chains such as β-1,6-galactohexaose (β-1,6-Gal6) also exist in type II AGs. To gain insight into the structures important for AGP functions, in vivo structural modification of β-1,6-galactan side chains was performed in Arabidopsis. We generated transgenic Arabidopsis plants expressing a fungal endo-β-1,6-galactanase, Tv6GAL, that degrades long side chains specifically under the control of dexamethasone (Dex). Two of 6 transgenic lines obtained showed more than 40 times activity of endo-β-1,6-galactanase when treated with Dex. Structural analysis indicated that long side chains such as β-1,6-Gal5 and β-1,6-Gal6 were significantly reduced compared to wild-type plants. Tv6GAL induction caused retarded growth of seedlings, which had a reduced amount of cellulose in cell walls. These results suggest that long β-1,6-galactan side chains are necessary for normal cellulose synthesis and/or deposition as their defect affects cell growth in plants.

Abstract 13606: Deletion of CD47 Specifically in Lymphatic Endothelium Augments Arterial Lymphangiogenesis and Attenuates Atherosclerosis

Thrombospondin-1 (TSP1), an extracellular glycoprotein mediates differential physio- or pathological effects via interacting with cell surface receptors including CD36 and CD47. TSP1 is a well-known angiogenesis inhibitor, however, its role in regulating lymphangiogenesis is not clear. We recently reported that global CD47-deficient mice are protected from atherosclerosis, however, myeloid cell-specific CD47 loss augments lesion formation suggesting the importance of cell-specific CD47 blockade in atherosclerosis. Our experiments revealed CD47 as a dominant TSP1 receptor in lymphatic endothelial cells (LECs). As the lymphatic vasculature is functionally linked to atherosclerosis, we herein investigated the effects of LEC TSP1-CD47 signaling inhibition on lymphangiogenesis and atherosclerotic lesion formation. Our immunoblotting data demonstrated elevated TSP1 expression in human and mouse atherosclerotic aortic tissue compared to non-atherosclerotic control tissue. TSP1 at pathological concentrations inhibited VEGF-C-stimulated in vitro lymphangiogenesis. Mechanistically, TSP1 inhibited VEGF-C-induced Akt activation in LECs, leading to decreased eNOS phosphorylation and attenuated nitric oxide production. Further, TSP1-treated CD47-silenced LECs proliferate faster and have higher Akt and eNOS activation than TSP1-treated control cells. AAV8-PCSK9-injected LEC-specific CD47 knockout ( Cd47 f/f Lyve-1 Cre +/- ) mice had reduced atherosclerosis in both aorta and aortic root compared with control ( Cd47 f/f ) mice. Additional immunostaining experiments performed on aortic root cross-sections indicated improved lymphatic vessel density in Cd47 f/f Lyve-1 Cre +/- mice. These results demonstrate that TSP1-mediated LEC CD47 activation inhibits lymphangiogenesis and blockade of LEC CD47 signaling suppresses atherosclerosis. Altogether, these findings identify LEC CD47 as a potential therapeutic target in atherosclerosis.