Biochemistry, Biophysics, and Structural Biology Commons^™

Role Of The Hypervariable Hinge Region Of Phosphoprotein P Of Vesicular Stomatitis Virus In Viral Rna Synthesis And Assembly Of Infectious Virus Particles, Subash C. Das, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The phosphoprotein (P protein) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNAdependent RNA polymerase and has multiple functions residing in its different domains. In the present study, we examined the role of the hypervariable hinge region of P protein in viral RNA synthesis and recovery of infectious VSV by using transposon-mediated insertion mutagenesis and deletion mutagenesis. We observed that insertions of 19-amino-acid linker sequences at various positions within this region affected replication and transcription functions of the P protein to various degrees. Interestingly, one insertion mutant was completely defective in both transcription and replication. Using …

Crystallization And Preliminary X-Ray Analysis Of Na-Asp-1, A Multi-Domain Pathogenesis-Related-1 Protein From The Human Hookworm Parasite Necator Americanus, Oluwatoyin A. Asojo, Alex Loukas, Mehmet Inan, Rick Barent, Jicai Huang, Brad Plantz, Amber Swanson, Mark Gouthro, Michael M. Meagher, Peter J. Hotez

Department of Chemical and Biomolecular Engineering: Faculty Publications

Human hookworm infection is a major cause of anemia and malnutrition in the developing world. In an effort to control hookworm infection, the Human Hookworm Vaccine Initiative has identified candidate vaccine antigens from the infective larval stage (L3) of the parasite, including a family of pathogenesis-related-1 (PR-1) proteins known as the ancylostoma-secreted proteins (ASPs). The functions of the ASPs are unknown. In addition, it is unclear why some ASPs have one while others have multiple PR-1 domains. There are no known structures of a multi-domain ASP and in an effort to remedy this situation, recombinant Na-ASP-1 has been expressed, …

An Algorithm For Identification Of Bacterial Selenocysteine Insertion Sequence Elements And Selenoprotein Genes, Yan Zhang, Vadim Gladyshev

Department of Biochemistry: Faculty Publications

Motivation: Incorporation of selenocysteine (Sec) into proteins in response to UGA codons requires a cis-acting RNA structure, Sec insertion sequence (SECIS) element. Whereas SECIS elements in Escherichia coli are well characterized, a bacterial SECIS consensus structure is lacking.

Results: We developed a bacterial SECIS consensus model, the key feature of which is a conserved guanosine in a small apical loop of the properly positioned structure. This consensus was used to build a computational tool, bSECISearch, for detection of bacterial SECIS elements and selenoprotein genes in sequence databases. The program identified 96.5% of known selenoprotein genes in completely sequenced bacterial genomes …

Ph-Dependent Substrate Preference Of Pig Heart Lipoamide Dehydrogenase Varies With Oligomeric State: Response To Mitochondrial Matrix Acidification, Natalia L. Klyachko, Valentina A. Shchedrina, Alexander V. Efimov, Sergey V. Kazakov, Irina G. Gazaryan, Bruce S. Kristal, Abraham M. Brown

Department of Biochemistry: Faculty Publications

Cycling of intracellular pH has recently been shown to play a critical role in ischemia-reperfusion injury. Ischemia-reperfusion also leads to mitochondrial matrix acidification and dysfunction. However, the mechanism by which matrix acidification contributes to mitochondrial dysfunction, oxidative stress, and the resultant cellular injury has not been elucidated. We observe pH-dependent equilibria between monomeric, dimeric, and a previously undescribed tetrameric form of pig heart lipoamide dehydrogenase (LADH), a mitochondrial matrix enzyme. Dynamic light scattering studies of native LADH in aqueous solution indicate that lowering pH favors a shift in average molecular mass from higher oligomeric states to monomer. Sedimentation velocity of …

Regulation Of Enteric Endophytic Bacterial Colonization By Plant Defenses, A. Leonardo Iniguez, Yuemei Dong, Heather D. Carter, Brian M. M. Ahmer, Julie M. Stone, Eric W. Triplett

Department of Biochemistry: Faculty Publications

Bacterial endophytes reside within the interior of plants without causing disease or forming symbiotic structures. Some endophytes, such as Klebsiella pneumoniae 342 (Kp342), enhance plant growth and nutrition. Others, such as Salmonella enterica serovar Typhimurium (S. typhimurium), are human pathogens that contaminate raw produce. Several lines of evidence are presented here to support the hypothesis that plant defense response pathways regulate colonization by endophytic bacteria. An ethyleneinsensitive mutant of Medicago truncatula is hypercolonized by Kp342 compared to the parent genotype. Addition of ethylene, a signal molecule for induced systemic resistance in plants, decreased endophytic colonization in Medicago spp. …

Phylogenetic Engineering At An Interface Between Large And Small Subunits Imparts Land-Plant Kinetic Properties To Algal Rubisco, Robert J. Spreitzer, Srinivasa R. Peddi, Sriram Satagopan

Department of Biochemistry: Faculty Publications

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO₂ fixation and, thus, limits agricultural productivity. However, Rubisco enzymes from different species have different catalytic constants. If the structural basis for such differences were known, a rationale could be developed for genetically engineering an improved enzyme. Residues at the bottom of the large-subunit α/β-barrel active site of Rubisco from the green alga Chlamydomonas reinhardtii (methyl-Cys-256, Lys-258, and Ile-265) were previously changed through directed mutagenesis and chloroplast transformation to residues characteristic of land-plant Rubisco (Phe-256, Arg-258, and Val-265). The resultant enzyme has decreases in carboxylation efficiency and CO₂/O …

Roles For Nutrients In Epigenetic Events, Anna M. Oommen, Jacob B. Griffin, Gautam Sarath, Janos Zempleni

Gautam Sarath Publications

The field of epigenetics is the study of modifications of DNA and DNA-binding proteins that alter the structure of chromatin without altering the nucleotide sequence of DNA; some of these modifications may be associated with heritable changes in gene function. Nutrients play essential roles in the following epigenetic events. First, folate participates in the generation of S-adenosylmethionine, which acts as a methyl donor in the methylation of cytosines in DNA; methylation of cytosines is associated with gene silencing. Second, covalent attachment of biotin to histones (DNA-binding proteins) plays a role in gene silencing and in the cellular response to DNA …

Imposed Constraints On The Smith-Waterman Alignment Algorithm For Enhanced Modeling Of A Single-Molecule Dna Sequencer, Patrick G. Humphrey, Gregory R. Bashford

Biomedical Imaging and Biosignal Analysis Laboratory

An effort has been underway to develop a system for de novo sequencing of single DNA molecules with very long reads. The system operates by optically detecting the passage of fluorescently tagged DNA bases through a detection zone. A successful system would be revolutionary with respect to speed, read length, cost and minimized laboratory infrastructure. An important part of system development is modeling of the detection process. In particular, predicting the expected error from a set of sequencing parameters is helpful in system design. This paper describes variations on the Smith-Waterman algorithm for subsequence alignment used in a single-molecule detection …

Modeling The Tertiary Structure Of A Maize (Zea Mays Ssp. Mays) Non-Symbiotic Hemoglobin, Juán Sáenz-Rivera, Gautam Sarath, Raul Peter-Arredondo

Gautam Sarath Publications

The tertiary structure of a maize (Zea mays ssp. mays) non-symbiotic hemoglobin (Hbm) was modeled using computer tools and the known tertiary structure of rice Hb1 as a template. This method was tested by predicting the tertiary structure of soybean leghemoglobin a (Lba) using rice Hb1 as a template. The tertiary structures of the predicted and native Lba were similar, indicating that our computer methods could reliably predict the tertiary structures of plant Hbs. We next predicted the tertiary structure of Hbm. Hbm appears to have a long pre-helixA and a large CD-loop. The positions of the distal …

Waccnes Containing Bovine Herpe Svirus 1 Attenuated By Mutation In Latency-Related Gene, Clinton J. Jones

School of Veterinary and Biomedical Sciences: Faculty Publications

Vaccines for pathogenic Strains of bovine herpesvirus 1 (BHV-1) which are based on attenuated BHV-1 having a mutation in the latency-related gene are provided. Live, attenuated vaccines are also provided which express anti gens from other viral or bacterial pathogens and thus form the basis of a variety of vaccines.

Identification And Characterization Of Phosphoseryl-Trna [Ser]Sec Kinase, Bradley A. Carlson, Xue-Ming Xu, Gregory V. Kryukov, Mahadev Rao, Marla J. Berry, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

In 1970, a kinase activity that phosphorylated a minor species of seryl-tRNA to form phosphoseryl-tRNA was found in rooster liver [Maenpaa, P. H. & Bernfield, M. R. (1970) Proc. Natl. Acad. Sci. USA 67, 688–695], and a minor seryl-tRNA that decoded the nonsense UGA was detected in bovine liver. The phosphoseryl-tRNA and the minor UGA-decoding seryl-tRNA were subsequently identified as selenocysteine (Sec) tRNA ^[Ser]Sec, but the kinase activity remained elusive. Herein, by using a comparative genomics approach that searched completely sequenced archaeal genomes for a kinase-like protein with a pattern of occurrence similar to that of components of Sec …

Flash Artifact Suppression In Two-Dimensional Ultrasound Imaging, Richard Yung Chiao, Gregory Ray Bashford, Mark Peter Feilen, Cynthia Andrews Owen

Biomedical Imaging and Biosignal Analysis Laboratory

Flash artifacts in ultrasound flow images are suppressed to achieve enhanced flow discrimination. Flash artifacts typically occur as region of elevated signal strength (brightness or equivalent color) within an image. A flash suppression algorithm included the steps of estimating the flash within an image and then suppressing the estimated flash. The mechanism for flash suppression is spatial filtering. An extension of this basic method used information from adjacent frames to estimate the flash and/or to smooth the resulting image sequence. Temporal information from adjacent frames is used as an adjunct to improve performance.

Hyaluronan Facilitates Invasion Of Colon Carcinoma Cells In Vitro Via Interaction With Cd44, Hyeong-Rok Kim, Marie A. Wheeler, Christopher M. Wilson, Joji Iida, David Eng, Melanie A. Simpson, James B. Mccarthy, Kelli M. Bullard

Department of Biochemistry: Faculty Publications

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in …

Melanoma Chondroitin Sulfate Proteoglycan Enhances Fak And Erk Activation By Distinct Mechanisms, Jianbo Yang, Matthew A. Price, Cheryl L. Neudauer, Christopher Wilson, Soldano Ferrone, Hong Xia, Joji Iida, Melanie A. Simpson, James B. Mccarthy

Department of Biochemistry: Faculty Publications

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal–regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes …

Characterization Of Human Udp-Glucose Dehydrogenase: Cys-276 Is Required For The Second Of Two Successive Oxidations, Brandi J. Sommer, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for K_m and V_max that agree well with those reported for a bovine homolog. We used crystal coordinates …

The Prokaryotic Selenoproteome, Gregory V. Kryukov, Vadim N. Gladyshev

Vadim Gladyshev Publications

In the genetic code, the UGA codon has a dual function as it encodes selenocysteine (Sec) and serves as a stop signal. However, only the translation terminator function is used in gene annotation programs, resulting in misannotation of selenoprotein genes. Here, we applied two independent bioinformatics approaches to characterize a selenoprotein set in prokaryotic genomes. One method searched for selenoprotein genes by identifying RNA stem–loop structures, selenocysteine insertion sequence elements; the second approach identified Sec/Cys pairs in homologous sequences. These analyses identified all or almost all selenoproteins in completely sequenced bacterial and archaeal genomes and provided a view on the …

Specific Excision Of The Selenocysteine Trna [Ser]Sec (Trsp) Gene In Mouse Liver Demonstrates An Essential Role Of Selenoproteins In Liver Function, Bradley A. Carlson, Sergey V. Novoselov, Easwari Kumaraswamy, Byeong Jae Lee, Miriam R. Anver, Vadim N. Gladyshev, Dolph L. Hatfield

Vadim Gladyshev Publications

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA ^[Ser]Sec gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA …

The Identity Of Proteins Associated With A Small Heat Shock Protein During Heat Stress In Vivo Indicates That These Chaperones Protect A Wide Range Of Cellular Functions, Eman Basha, Garrett J. Lee, Linda A. Breci, Andrew C. Hausrath, Nicole R. Buan, Kim C. Giese, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen …

Methionine Sulfoxide Reduction In Mammals: Characterization Of Methionine-R-Sulfoxide Reductases, Hwa-Young Kim, Vadim Gladyshev

Vadim Gladyshev Publications

Methionine residues in proteins are susceptible to oxidation by reactive oxygen species, but can be repaired via reduction of the resulting methionine sulfoxides by methionine-S-sulfoxide reductase (MsrA) and methionine-R-sulfoxide reductase (MsrB). However, the identity of all methionine sulfoxide reductases involved, their cellular locations and relative contributions to the overall pathway are poorly understood. Here, we describe a methionine-R-sulfoxide reduction system in mammals, in which two MsrB homologues were previously described. We found that human and mouse genomes possess three MsrB genes and characterized their protein products, designated MsrB1, MsrB2, and MsrB3. MsrB1 (Selenoprotein R) was present in the cytosol and …

Tissue-Specific Expression And Developmental Regulation Of Cytochrome B561 Genes In Arabidopsis Thaliana And Raphanus Sativus, Wim Verelst, Jyoti Kapila, Janice De Almeida Engler, Julie M. Stone, Roland Caubergs, Han Asard

Department of Biochemistry: Faculty Publications

Ascorbate (Asc) is an essential molecule in many aspects of development and stress responses in plants and animals. Cytochromes b561 (cyts b561) are tightly coupled to Asc homeostasis. These proteins are found in mammalian tissues, where they are involved in the regeneration of Asc, serving the synthesis of catecholamine neurotransmitters, and in intestinal iron reduction. Plant genomes encode homologous membrane-associated, Asc-reducible cyts b561. The expression of these proteins in plants, however, has so far not been studied. We have now examined the expression of two Arabidopsis thaliana cyt b561-encoding genes—Artb561-1 and Artb561-2—using relative-quantitative RT-PCR …

Identification Of Trace Element-Containing Proteins In Genomic Databases, Vadim N. Gladyshev, Gregory V. Kryukov, Dmitri E. Fomenko, Dolph L. Hatfield

Vadim Gladyshev Publications

Development of bioinformatics tools provided researchers with the ability to identify full sets of trace element–containing proteins in organisms for which complete genomic sequences are available. Recently, independent bioinformatics methods were used to identify all, or almost all, genes encoding selenocysteine-containing proteins in human, mouse, and Drosophila genomes, characterizing entire selenoproteomes in these organisms. It also should be possible to search for entire sets of other trace element–associated proteins, such as metal-containing proteins, although methods for their identification are still in development.

Hospital Universitario, “La Paz”, Madrid, Spain, Michael G. Zeece, Gautam Sarath, John P. Markwell, N. Palmer, J. Steckelberg, J. Schultz

Gautam Sarath Publications

RATIONALE: Proteomic technology has been shown to have distinct advantages for allergen characterization and identification. Therefore, we have used 2-D electrophoresis and protein microarrays to characterize the profile of allergens and assess their level in lentils.

METHODS: Lentil varieties were extracted and centrifuged to obtain 2S, 7S and 11S fractions. The profile of IgE-binding proteins in these fractions was assessed using lentil-allergic sera, by immunoblotting of 2-D electrophoretic separations. The level of IgE-binding was also assessed using protein microarrays. Arrays of covalently attached proteins were first incubated with lentil-allergic sera, followed by incubation with fluorescently- labeled anti-IgE detection antibody. Laser-induced …

Phosphorylation Of Vesicular Stomatitis Virus Phosphoprotein P Is Indispensable For Virus Growth†, Subash C. Das, Asit K. Pattnaik

School of Veterinary and Biomedical Sciences: Faculty Publications

The phosphoprotein (P) of vesicular stomatitis virus (VSV) is an essential subunit of the viral RNAdependent RNA polymerase (RdRp) complex. It is phosphorylated at two different domains. Using defective interfering (DI) RNA or minigenomic RNA templates, we previously demonstrated that phosphorylation within the amino-terminal domain I is essential for transcription, whereas phosphorylation within the carboxyterminal domain II is necessary for replication. For the present study, we examined the role of the phosphorylation of residues in these domains in the life cycle of VSV. Various mutant P coding sequences were inserted into a full-length cDNA clone of VSV, and the virus …

Interactions Between Small Heat Shock Protein Subunits And Substrate In Small Heat Shock Protein-Substrate Complexes, Kenneth L. Friedrich, Kim C. Giese, Nicole R. Baun, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

Small heat shock proteins (sHSPs) are dynamic oligomeric

proteins that bind unfolding proteins and protect

them from irreversible aggregation. This binding results

in the formation of sHSP-substrate complexes from

which substrate can later be refolded. Interactions between

sHSP and substrate in sHSP-substrate complexes

and the mechanism by which substrate is transferred to

ATP-dependent chaperones for refolding are poorly defined.

We have established C-terminal affinity-tagged

sHSPs from a eukaryote (pea HSP18.1) and a prokaryote

(Synechocystis HSP16.6) as tools to investigate these issues.

We demonstrate that sHSP subunit exchange for

HSP18.1 and HSP16.6 is temperature-dependent and

rapid at the optimal growth …

Methionine Sulfoxide Reductase Regulation Of Yeast Lifespan Reveals Reactive Oxygen Species-Dependent And -Independent Components Of Aging, Ahmet Koc, Audrey P. Gasch, Julian C. Rutherford, Hwa-Young Kim, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Aging is thought to be caused by the accumulation of damage, primarily from oxidative modifications of cellular components by reactive oxygen species (ROS). Here we used yeast methionine sulfoxide reductases MsrA and MsrB to address this hypothesis. In the presence of oxygen, these antioxidants could increase yeast lifespan and did so independent of the lifespan extension offered by caloric restriction. However, under ROS-deficient, strictly anaerobic conditions, yeast lifespan was shorter, not affected by MsrA or MsrB, and further reduced by caloric restriction. In addition, we identified changes in the global gene expression associated with aging in yeast, and they did …

The Identity Of Proteins Associated With A Small Heat Shock Protein During Heat Stress In Vivo Indicates That These Chaperones Protect A Wide Range Of Cellular Functions, Eman Basha, Garrett J. Lee, Linda A. Breci, Andrew C. Hausrath, Nicole R. Baun, Kim C. Giese, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

The small heat shock proteins (sHSPs) are a ubiquitous

class of ATP-independent chaperones believed to

prevent irreversible protein aggregation and to facilitate

subsequent protein renaturation in cooperation

with ATP-dependent chaperones. Although sHSP chaperone

activity has been studied extensively in vitro, understanding

the mechanism of sHSP function requires

identification of proteins that are sHSP substrates in

vivo. We have used both immunoprecipitation and affinity

chromatography to recover 42 proteins that specifically

interact with Synechocystis Hsp16.6 in vivo during

heat treatment. These proteins can all be released from

Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones

and are heat-labile. …

Reconsidering The Evolution Of Eukaryotic Selenoproteins: A Novel Nonmammalian Family With Scattered Phylogenetic Distribution, Sergi Castellano, Sergey V. Novoselov, Gregory V. Kryukov, Alain Lescure, Enrique Blanco, Alain Krol, Vadim N. Gladyshev, Roderic Guigo

Vadim Gladyshev Publications

While the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome-wide prediction of genes with in-frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteinecontaining form also occurred in …

Reconsidering The Evolution Of Eukaryotic Selenoproteins: A Novel Nonmammalian Family With Scattered Phylogenetic Distribution, Sergi Castellano, Sergey V. Novoselov, Gregory V. Kryukov, Alain Lescure, Enrique Blanco, Alain Krol, Vadim N. Gladyshev, Roderic Guigo

Vadim Gladyshev Publications

While the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome-wide prediction of genes with in-frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteinecontaining form also occurred in …

A Role For Talin In Presynaptic Function, Jennifer R. Morgan, Gilbert Di Paolo, Hauke Werner, Valentina A. Shchedrina, Marc Pypaert, Vincent A. Pieribone, Pietro De Camilli

Department of Biochemistry: Faculty Publications

Talin, an adaptor between integrin and the actin cytoskeleton at sites of cell adhesion, was recently found to be present at neuronal synapses, where its function remains unknown. Talin interacts with phosphatidylinositol-(4)-phosphate 5-kinase type Iγ, the major phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]–synthesizing enzyme in brain. To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin–PIP kinase interaction and then examined their effects on synaptic structure. A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed. The endocytic defect included an accumulation of clathrin-coated pits …

Endocytic Function, Glycosaminoglycan Specificity, And Antibody Sensitivity Of The Recombinant Human 190-Kda Hyaluronan Receptor For Endocytosis (Hare), Ed Harris, Janet A. Weigel, Paul H. Weigel

Department of Biochemistry: Faculty Publications

The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339–349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (190hHARE cells). Like the native protein, recombinant hHARE contains ~25 kDa of N-linked oligosaccharides, binds HA in a ligand blot …