Combined biochemical and cytological analysis of membrane trafficking using lectins

Human immunodeficiency virus -1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons.

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“…et. al., 2003; Sitia, R. et al, 2003; Ohtsubo et al, 2006; Aebi M. et al, 2010); Morgan G.W. et al, 2013).…”

Section: Introductionunclassified

Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons

Teodorof

Divakar

Soontornniyomkij

et al. 2014

Neurobiology of Disease

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“…The endoplasmic reticulum was stained with tetramethylrhodamine-labeled concanavalin A (Invitrogen) in permeabilization solution without serum. 33 After washing, coverslips were transferred to the slides and maintained in fluorescent mounting medium (DakoCytomation, Glostrup, Denmark). Slides were analyzed using a Nikon Eclipse Ti confocal microscope (Nikon Corp., Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Lymphocyte phosphatase‐associated phosphoprotein proteoforms analyzed using monoclonal antibodies

2015

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Phosphatase CD45 regulates the activation of lymphocytes by controlling the level of receptor and signal molecule phosphorylation. However, it remains unknown which molecules mediate the phosphatase activity of CD45. A candidate for such a molecule is a small transmembrane adapter protein called lymphocyte phosphatase-associated phosphoprotein (LPAP). LPAP forms a supramolecular complex that consists of not only CD45 molecule but also CD4 and Lck kinase. The function of LPAP has not been defined clearly. In our study, we determined the pattern of LPAP expression in various cell types and characterized its proteoforms using new monoclonal antibodies generated against the intracellular portion of the protein. We show that LPAP is a pan-lymphocyte marker, and its expression in cells correlates with the expression of CD45. The majority of T, B and NK cells express high levels of LPAP, whereas monocytes, granulocytes, monocyte-derived dendritic cells, platelets and red blood cells are negative for LPAP. Using one- and two-dimensional protein gel electrophoresis, we demonstrate that LPAP has at least four sites of phosphorylation. The resting cells express at least six different LPAP phosphoforms representing mono-, di- and tri-phosphorylated LPAP. T and B cells differ in the distribution of the protein between phosphoforms. The activation of lymphocytes with PMA reduces the diversity of phosphorylated forms. Our experiments on Lck-deficient Jurkat cells show that Lck kinase is not involved in LPAP phosphorylation. Thus, LPAP is a dynamically phosphorylated protein, the function of which can be understood, when all phosphosites and kinases involved in its phosphorylation will be identified.

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“…These well‐labeled lectins are also very powerful tools for the study of membrane trafficking . For example, redistribution of Galanthus nivalis agglutinin (GNA) binding glycoproteins triggered by guanosine 5′‐O‐(3‐thio) triphosphate (GTPγS), which perturbs endoplasmic reticulum (ER) Golgi transport, was tracked by GNA . It is interesting to find that after treatment of GTPγS, GNA‐containing vesicles tended to accumulate from ER to the perinuclear region of the cells .…”

Section: The Development Of Lectins As Biological Toolsmentioning

confidence: 99%

“…For example, redistribution of Galanthus nivalis agglutinin (GNA) binding glycoproteins triggered by guanosine 5′‐O‐(3‐thio) triphosphate (GTPγS), which perturbs endoplasmic reticulum (ER) Golgi transport, was tracked by GNA . It is interesting to find that after treatment of GTPγS, GNA‐containing vesicles tended to accumulate from ER to the perinuclear region of the cells . The application of GS‐II‐Alexa 594 or GNL‐Alexa 647 conjugate together with other methods to the characterization of conserved oligomeric Golgi (COG) depleted cells revealed the importance of COG complex in trafficking and maintenance of glycosylation machinery .…”

Section: The Development Of Lectins As Biological Toolsmentioning

confidence: 99%

“…Lectin conjugation methods described in the “Lectin histochemistry and cytochemistry” section could also be applied here . After electrophoresis, proteins were transferred to nitrocellulose membranes, equilibrated and incubated with biotinylated lectins, followed by washing and incubation with streptavidin–alkaline phosphatase conjugate . The blots will then be developed with BCI/NBT (5‐bromo‐4‐chloro‐3‐indolyphosphate/nitro blue tetrazolium) substrate .…”

Section: The Development Of Lectins As Biological Toolsmentioning

confidence: 99%

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Development and Applications of Lectins as Biological Tools in Biomedical Research

Dan

Liu

2015

Medicinal Research Reviews

108

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As a new and burgeoning area following genomics and proteomics, glycomics has become a hot issue due to its pivotal roles in many physiological and pathological processes. Glycans are much more complicated than genes or proteins since glycans are highly branched and dynamic. Antibodies and lectins are the two major molecular tools applied for glycan profiling. Though the study of antibodies and lectins started at almost the same time in 1880s, lectins gained much less attention than the antibodies until recent decades when the importance and difficulties of glycomics were realized. The present review summarizes the discovery history of lectins and their biological functions with a special emphasis on their various applications as biological tools. Both older techniques that had been developed in the last century and new technologies developed in recent years, especially lectin microarrays and lectin-based biosensors, are included in this account.

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Combined biochemical and cytological analysis of membrane trafficking using lectins

Cited by 9 publications

References 57 publications

Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons

Intracellular mannose binding lectin mediates subcellular trafficking of HIV-1 gp120 in neurons

Lymphocyte phosphatase‐associated phosphoprotein proteoforms analyzed using monoclonal antibodies

Development and Applications of Lectins as Biological Tools in Biomedical Research

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