scholarly journals Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

2012 ◽  
Vol 287 (42) ◽  
pp. 35658-35668 ◽  
Author(s):  
Sung E. Kwon ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Post Translational Modification ◽  
Vesicle Membrane ◽  
Knock Out ◽  
Synaptic Localization ◽  
Glycosylation Sites ◽  
Synaptotagmin 1 ◽  
Knock Out Mice ◽  
And Function

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.

Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and disease

2004 ◽  
Vol 82 (1) ◽  
pp. 129-144 ◽  
Author(s):  
Andrew F.G Quest ◽  
Lisette Leyton ◽  
Mario Párraga
Keyword(s):  
Lipid Rafts ◽  
Structural Integrity ◽  
Membrane Microdomains ◽  
Cellular Transport ◽  
Knock Out ◽  
Caveolin 1 ◽  
Membrane Compartments ◽  
Knock Out Mice ◽  
And Function

Caveolae were initially described some 50 years ago. For many decades, they remained predominantly of interest to structural biologists. The identification of a molecular marker for these domains, caveolin, combined with the possibility to isolate such cholesterol- and sphingolipid-rich regions as detergent-insoluble membrane complexes paved the way to more rigorous characterization of composition, regulation, and function. Experiments with knock-out mice for the caveolin genes clearly demonstrate the importance of caveolin-1 and -3 in formation of caveolae. Nonetheless, detergent-insoluble domains are also found in cells lacking caveolin expression and are referred to here as lipid rafts. Caveolae and lipid rafts were shown to represent membrane compartments enriched in a large number of signaling molecules whose structural integrity is essential for many signaling processes. Caveolin-1 is an essential structural component of cell surface caveolae, important for regulating trafficking and mobility of these vesicles. In addition, caveolin-1 is found at many other intracellular locations. Variations in subcellular localization are paralleled by a plethora of ascribed functions for this protein. Here, more recent data addressing the role of caveolin-1 in cellular signaling and the development of diseases like cancer will be preferentially discussed.Key words: caveolae, rafts, membrane microdomains, caveolins, signal transduction, disease, cancer.

Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease

2005 ◽  
Vol 94 (09) ◽  
pp. 471-487 ◽  
Author(s):  
Judith Leurs ◽  
Dirk Hendriks
Keyword(s):  
Bleeding Tendency ◽  
Knock Out ◽  
Lysine Residues ◽  
Knock Out Mice ◽  
The Moment ◽  
And Function ◽  
Coagulation And Fibrinolysis

SummarySince the discovery of Carboxypeptidase U (CPU) in 1988, considerable information has been gathered about its biochemistry and function in physiological and pathophysiological circumstances. A variety of tools such as assays to measure proCPU and CPU, antibodies raised against (pro)CPU, selective CPU inhibitors and knock-out mice have been developed and are currently being used to explore the role of this metallocarboxypeptidase in different in vivo and in vitro settings. The knowledge that proCPU can be activated by thrombin and plasmin, enzymes with a key function in coagulation and fibrinolysis, and the ability of CPU to remove C-terminal lysine residues has led to the hypothesis that the proCPU/CPU pathway plays a role in the balance between coagulation and fibrinolysis. The maintenance of the equilibrium between coagulation and fibrinolysis is crucial for normal haemostasis and disturbance of this delicate balance can lead either to bleeding tendency or thrombosis. This review provides an update on several aspects of CPU known at the moment, including an extensive overview on the clinical studies performed up till now.J. Leurs is a research assistant of the Fund for Scientific Research Flanders (FWO-Vlaanderen).

Endothelial Tyrosine Kinase Tie1 Is Required for Normal Schlemm’s Canal Development

2022 ◽  
Author(s):  
Jing Du ◽  
Benjamin R. Thomson ◽  
Tuncer Onay ◽  
Susan E. Quaggin
Keyword(s):  
Intraocular Pressure ◽  
Quantitative Polymerase Chain Reaction ◽  
Schlemm’S Canal ◽  
Knock Out ◽  
Schlemm's Canal ◽  
Elevated Intraocular Pressure ◽  
Knock Out Mice ◽  
And Function ◽  
The Impact

Background: Schlemm’s canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. Methods: We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. Results: Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. Conclusions: Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

scholarly journals Loss of microglial SIRPα promotes synaptic pruning in preclinical models of neurodegeneration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xin Ding ◽  
Jin Wang ◽  
Miaoxin Huang ◽  
Zhangpeng Chen ◽  
Jing Liu ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Knock Out ◽  
Classical Complement Pathway ◽  
Synaptic Remodeling ◽  
The Central Nervous System ◽  
System Activation ◽  
Knock Out Mice ◽  
Synaptic Pruning

AbstractMicroglia play a key role in regulating synaptic remodeling in the central nervous system. Activation of classical complement pathway promotes microglia-mediated synaptic pruning during development and disease. CD47 protects synapses from excessive pruning during development, implicating microglial SIRPα, a CD47 receptor, in synaptic remodeling. However, the role of microglial SIRPα in synaptic pruning in disease remains unclear. Here, using conditional knock-out mice, we show that microglia-specific deletion of SIRPα results in decreased synaptic density. In human tissue, we observe that microglial SIRPα expression declines alongside the progression of Alzheimer’s disease. To investigate the role of SIRPα in neurodegeneration, we modulate the expression of microglial SIRPα in mouse models of Alzheimer’s disease. Loss of microglial SIRPα results in increased synaptic loss mediated by microglia engulfment and enhanced cognitive impairment. Together, these results suggest that microglial SIRPα regulates synaptic pruning in neurodegeneration.

scholarly journals REST is a major negative regulator of endocrine differentiation during pancreas organogenesis

2021 ◽  
Author(s):  
Meritxell Rovira ◽  
Goutham Atla ◽  
Miguel Angel Maestro ◽  
Vane Grau ◽  
Javier García-Hurtado ◽  
...  
Keyword(s):  
Endocrine Cells ◽  
Negative Regulator ◽  
Β Cell ◽  
Knock Out ◽  
Endocrine Differentiation ◽  
Conditional Allele ◽  
Embryonic Pancreas ◽  
Pancreatic Endocrine Cells ◽  
Knock Out Mice

SUMMARYUnderstanding genomic regulatory mechanisms of pancreas differentiation is relevant to the pathophysiology of diabetes mellitus, and to the development of replacement therapies. Numerous transcription factors promote β cell differentiation, although less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrine gene programs in the embryonic pancreas. However, pancreaticRestknock-out mice failed to show increased numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we now observe a marked increase in the formation of pancreatic endocrine cells. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts, and induced β-cell specific genes in human adult duct-derived organoids. Finally, we define REST genomic programs that suppress pancreatic endocrine differentiation. These results establish a crucial role of REST as a negative regulator of pancreatic endocrine differentiation.

scholarly journals Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH

2018 ◽  
Vol 92 (9) ◽  
pp. e00084-18 ◽  
Author(s):  
Melina Vallbracht ◽  
Sascha Rehwaldt ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Walter Fuchs
Keyword(s):  
Pseudorabies Virus ◽  
Viral Envelope ◽  
Fusion Activity ◽  
Virus Particles ◽  
Glycosylation Sites ◽  
Functional Relevance ◽  
And Function ◽  
Cell Spread

ABSTRACTMany viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reducedin vitrofusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in theVaricellovirusgenus, resulted in enhancedin vitrofusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.IMPORTANCEHerpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport,in vitrofusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

scholarly journals Anatomical, Physiological, and Pharmacological Characteristics of Histidine Decarboxylase Knock-Out Mice: Evidence for the Role of Brain Histamine in Behavioral and Sleep–Wake Control

2002 ◽  
Vol 22 (17) ◽  
pp. 7695-7711 ◽  
Author(s):  
Régis Parmentier ◽  
Hiroshi Ohtsu ◽  
Zahia Djebbara-Hannas ◽  
Jean-Louis Valatx ◽  
Takehiko Watanabe ◽  
...  
Keyword(s):  
Histidine Decarboxylase ◽  
Brain Histamine ◽  
Knock Out ◽  
Knock Out Mice

Absence of orthogonal arrays in kidney, brain and muscle from transgenic knockout mice lacking water channel aquaporin-4

1997 ◽  
Vol 110 (22) ◽  
pp. 2855-2860 ◽  
Author(s):  
J.M. Verbavatz ◽  
T. Ma ◽  
R. Gobin ◽  
A.S. Verkman
Keyword(s):  
Cho Cells ◽  
Collecting Duct ◽  
Water Channel ◽  
Freeze Fracture ◽  
Orthogonal Arrays ◽  
Knock Out ◽  
Orthogonal Arrays Of Particles ◽  
Freeze Fracture Electron Microscopy ◽  
Knock Out Mice ◽  
And Function

Freeze-fracture electron microscopy (FFEM) of kidney collecting duct, muscle, astrocytes in brain, and other mammalian tissues has revealed regular square arrays of intramembrane particles called orthogonal arrays of particles (OAPs). Their possible role in membrane structure and transport have been proposed, and their absence or decrease has been noted in a variety of hereditary and acquired diseases. A transgenic mouse lacking water channel AQP4 was used to show that AQP4 is the OAP protein. FFEM was done on kidney, skeletal muscle, and brain from AQP4 wild-type [+/+], heterozygous [+/−] and knock-out [-/-] mice. The [-/-] mice did not express detectable AQP4 protein, but were grossly indistinguishable from [+/+] mice. FFEM was done on blinded samples of kidney, brain and muscle from 9 mice. In all 6 kidney samples from [+/+] and [+/−] mice, OAPs similar to those in AQP4-transfected CHO cells were found in basolateral membranes of collecting duct principal cells. In all muscle and brain samples from [+/+] and [+/−] mice, OAPs of identical ultrastructure to those in kidney were seen, but in smaller patch sizes. OAPs were not seen in any sample from [-/-] mice. Label-fracture analysis using a peptide-derived AQP4 polyclonal antibody showed immunogold labeling of OAPs in AQP4-expressing CHO cells. These studies provide direct evidence that AQP4 is required for formation of OAPs and is a component of OAPs, thus establishing the identity and function of OAPs.

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