Studying Extracellular Signaling Utilizing a Glycoproteomic Approach: Lectin Blot Surveys, a First and Important Step

Cao, Jin; Guo, Shuzhen; Arai, Ken; Lo, Eng H.; Ning, MingMing

doi:10.1007/978-1-62703-426-5_15

Cited by 20 publications

(16 citation statements)

References 9 publications

(8 reference statements)

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“…En1-Cre; Srd5a3 fl/- mice were fertile and showed nearly Mendelian ratios at weaning age (data not shown). We used far-western blotting (far-WB) with biotinylated Sambucus Nigra lectin (SNA) to investigate the abundance of complex sialylated N-glycans ( Cao et al, 2013 ). Total protein extracts from mutant cerebellums showed a non-significant 12% decrease in normalized signal intensity ( Figure 1A,B ).…”

Section: Resultsmentioning

confidence: 99%

High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

Medina-Cano

Ucuncu

Nguyen

et al. 2018

eLife

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Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3, a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development.

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Section: Resultsmentioning

confidence: 99%

High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

Medina-Cano

Ucuncu

Nguyen

et al. 2018

eLife

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“…Compared with the method using various lectins to recognize different N-glycan chains and identify N-glycosylated proteins 42 , our method described here uses PNGase F to remove protein-linked N-glycan and detects the migration shift to identify the glycosylation of SCAP protein fragment a.a 540-707. The limitation of the lectin method is dependent upon the identification of the appropriate type of lectin to recognize the specific N-glycan.…”

Section: Discussionmentioning

confidence: 99%

“…To detect SCAP N-glycosylation in human cells or tissues, we need to develop a specific antibody against the amino acids 540 -707 fragment of human SCAP. In addition, identification of a specific lectin to recognize SCAP-bound N-glycan is an alternative way to analyze human SCAP N-glycosylation 42 . Moreover, defining the composition and structure of each N-glycan chain binding in SCAP (N263, N590 and N641) will facilitate our understanding of the underlying mechanism of SCAP trafficking from the ER to the Golgi.…”

Section: Discussionmentioning

confidence: 99%

Analysis of SCAP <em>N</em>-glycosylation and Trafficking in Human Cells

Cheng

Guo

Geng

et al. 2016

JoVE

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Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

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“…After lectin enrichment, more than a thousand proteins were identified to have glycosylation PTMs in the circulation. 101-103 Analysis of this rich network of signaling cascades with respect to clinical outcome and TH efficacy and side effects is under way. Thus, TH is also an ideal bedside stimulus-response model to study enzymatic and metabolic activity to gain direct insight into host response to injury and therapeutic efficacy and complications – crucial to the ultimate prognosis for the patient.…”

Section: Bedside Clinical Proteomic Profiling May Offer Insights mentioning

confidence: 99%

Pharmaco-proteomics opportunities for individualizing neurovascular treatment

Ning

Lopez

Sarracino

et al. 2013

Neurological Research

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Neurovascular disease often involves multi-organ system injury. For example, patent foramen ovale (PFO) related ischemic strokes involve not just the brain, but also the heart, the lung, and the peripheral vascular circulation. For higher-risk but high-reward systemic therapy (e.g., thrombolytics, therapeutic hypothermia, PFO closure) to be implemented safely, very careful patient selection and close monitoring of disease progression and therapeutic efficacy are imperative. For example, more than a decade after the approval of therapeutic hypothermic and intravenous thrombolysis treatments, they both remain extremely underutilized, in part due to lack of clinical tools for patient selection or to follow therapeutic efficacy. Therefore, in order to understand the complexity of the global effects of clinical neurovascular diseases and their therapies, a systemic approach may offer a unique perspective and provide tools with clinical utility. Clinical proteomic approaches may be promising to monitor systemic changes in complex multi-organ diseases – especially where the disease process can be “sampled” in clinically accessible fluid such as blood, urine and CSF. Here, we describe a “pharmaco-proteomic” approach to three major challenges in translational neurovascular research directly at bedside – in order to better stratify risk, widen therapeutic windows, explore novel targets to be validated at the bench – 1) thrombolytic treatment for ischemic stroke, 2) therapeutic hypothermia for post cardiac arrest syndrome, and 3) treatment for patent foramen ovale (PFO) related paradoxical embolic stroke. In the future, this clinical proteomics approach may help to improve patient selection, ensure more precise clinical phenotyping for clinical trials, and individualize patient treatment.

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Studying Extracellular Signaling Utilizing a Glycoproteomic Approach: Lectin Blot Surveys, a First and Important Step

Cited by 20 publications

References 9 publications

High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

Analysis of SCAP <em>N</em>-glycosylation and Trafficking in Human Cells

Pharmaco-proteomics opportunities for individualizing neurovascular treatment

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