Molecular cloning of a 47 kDa tissue-specific and differentiation-dependent urothelial cell surface glycoprotein

1993 ◽  
Vol 106 (1) ◽  
pp. 31-43 ◽  
Author(s):  
X.R. Wu ◽  
T.T. Sun
Keyword(s):  
Transmembrane Domain ◽  
Core Protein ◽  
Surface Glycoprotein ◽  
Peptide Sequence ◽  
Type I ◽  
Apical Surface ◽  
Bladder Epithelium ◽  
Cell Surface Glycoprotein ◽  
Signal Peptide Sequence ◽  
C Terminus

Despite the fact that bladder epithelium has many interesting biological features and is a frequent site of carcinoma formation, relatively little is known about its biochemical differentiation. We have shown recently that a 47 kDa glycoprotein, uroplakin III (UPIII), in conjunction with uroplakins I (27 kDa) and II (15 kDa), forms the asymmetric unit membrane (AUM)--a highly specialized biomembrane characteristic of the apical surface of bladder epithelium. Deglycosylation and cDNA sequencing revealed that UPIII contains up to 20 kDa of N-linked sugars attached to a core protein of 28.9 kDa. The presence of an N-terminal signal peptide sequence and a single transmembrane domain located near the C terminus, plus the N-terminal location of all the potential N-glycosylation sites, points to a type I (N-exo/C-cyto) configuration. Thus the mass of the extracellular domain (20 kDa plus up to 20 kDa of sugar) of UPIII greatly exceeds that of its intracellular domain (5 kDa). Such an asymmetrical mass distribution, a feature shared by the other two major uroplakins, provides a molecular explanation as to why the luminal leaflet of AUM is almost twice as thick as the cytoplasmic one. The fact that of the three major proteins of AUM only UPIII has a significant cytoplasmic domain suggests that this molecule may play an important role in AUM-cytoskeleton interaction in terminally differentiated urothelial cells.

scholarly journals A Cold-Sensitive Listeria monocytogenes Mutant Has a Transposon Insertion in a Gene Encoding a Putative Membrane Protein and Shows Altered (p)ppGpp Levels

2006 ◽  
Vol 72 (6) ◽  
pp. 3955-3959 ◽  
Author(s):  
Siqing Liu ◽  
Darrell O. Bayles ◽  
Tricia M. Mason ◽  
Brian J. Wilkinson
Keyword(s):  
Listeria Monocytogenes ◽  
Low Temperature ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Critical Role ◽  
Peptide Sequence ◽  
Temperature Adaptation ◽  
Signal Peptide Sequence ◽  
C Terminus ◽  
Cold Sensitive

ABSTRACT A cold-sensitive Listeria monocytogenes mutant designated cld-14 was obtained by transposon Tn917 mutagenesis. The gene interrupted by Tn917 in cld-14 was the L. monocytogenes LMOf2365_1485 homolog, which exhibits 45.7% homology to the Bacillus subtilis yqfF locus. LMOf2365_1485, here designated pgpH, encodes a putative integral membrane protein with a predicted molecular mass of 81 kDa. PgpH is predicted to contain a conserved N-terminal signal peptide sequence, seven transmembrane helices, and a hydrophilic C terminus, which likely extends into the cytosol. The Tn917 insertion in pgpH is predicted to result in production of a premature polypeptide truncated at the fifth transmembrane domain. The C terminus of PgpH, which is probably absent in cld-14, contains a highly conserved HD domain that belongs to a metal-dependent phosphohydrolase family. Strain cld-14 accumulated higher levels of (p)ppGpp than the wild type accumulated, indicating that the function of PgpH may be to adjust cellular (p)ppGpp levels during low-temperature growth. The cld-14pgpH + complemented strain was able to grow at a low temperature, like the parent strain, providing direct evidence that the activity of PgpH is important in low-temperature adaptation. Because of its predicted membrane location, PgpH may play a critical role in sensing the environmental temperature and altering cellular (p)ppGpp levels to allow the organism to adapt to low temperatures.

Cryptopain-1, a cysteine protease of Cryptosporidium parvum, does not require the pro-domain for folding

Parasitology ◽  
2008 ◽  
Vol 136 (2) ◽  
pp. 149-157 ◽  
Author(s):  
B.-K. NA ◽  
J.-M. KANG ◽  
H.-I. CHEUN ◽  
S.-H. CHO ◽  
S.-U. MOON ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Cysteine Protease ◽  
Transmembrane Domain ◽  
Cathepsin L ◽  
Active Form ◽  
Protozoan Parasite ◽  
Biochemical Properties ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Mature Domain

SUMMARYCryptosporidium parvum is an intracellular protozoan parasite that causes cryptosporidiosis in mammals including humans. In the current study, the gene encoding the cysteine protease of C. parvum (cryptopain-1) was identified and the biochemical properties of the recombinant enzyme were characterized. Cryptopain-1 shared common structural properties with cathepsin L-like papain family enzymes, but lacked a typical signal peptide sequence and contained a possible transmembrane domain near the amino terminus and a unique insert in the front of the mature domain. The recombinant cryptopain-1 expressed in Escherichia coli and refolded to the active form showed typical biochemical properties of cathepsin L-like enzymes. The folding determinant of cryptopain-1 was characterized through multiple constructs with or without different lengths of the pro-domain of the enzyme expressed in E. coli and assessment of their refolding abilities. All constructs, except one that did not contain the full-length mature domain, successfully refolded into the active enzymes, suggesting that cryptopain-1 did not require the pro-domain for folding. Western blot analysis showed that cryptopain-1 was expressed in the sporozoites and the enzyme preferentially degraded proteins, including collagen and fibronectin, but not globular proteins. This suggested a probable role for cryptopain-1 in host cell invasion and/or egression by the parasite.

Characterization of KIAA1427 protein as an atypical synaptotagmin (Syt XIII)

2001 ◽  
Vol 354 (2) ◽  
pp. 249-257 ◽  
Author(s):  
Mitsunori FUKUDA ◽  
Katsuhiko MIKOSHIBA
Keyword(s):  
Transmembrane Domain ◽  
Amino Acid Level ◽  
Subcellular Fractionation ◽  
Binding Activity ◽  
Type I ◽  
C2 Domains ◽  
C Terminus ◽  
Common Receptor ◽  
Antibody Uptake

Synaptotagmin (Syt) belongs to a family of type-I membrane proteins and is a protein that consists of a short extracellular N-terminus, a single transmembrane domain, two C2 domains and a short C-terminus. Here, we cloned and characterized a mouse orthologue of human KIAA1427 protein as an atypical Syt (named Syt XIII). Subcellular fractionation and antibody-uptake experiments indicate that Syt XIII is indeed a type-I membrane protein, but, unlike other Syt isoforms, lacks an N-terminal extracellular domain. Syt XIII C2 domains show relatively little similarity to Syt I (less than 35% identity at the amino acid level), and lack key amino acids responsible for Ca2+ binding. Because of these substitutions, the Syt XIII C2 domains did not show Ca2+-dependent phospholipid-binding activity, and Syt XIII is thus classified as a Ca2+-independent isoform. By contrast, the Syt XIII C-terminal domain is highly homologous with other Syt isoforms and can function as a common receptor for neurexin Iα in vitro. Since Syt XIII is expressed in various tissues outside the brain, Syt XIII may be involved in constitutive vesicle transport.

scholarly journals Influence of the Transposition of the Thermostabilizing Domain of Clostridium thermocellum Xylanase (XynX) on Xylan Binding and Thermostabilization

2002 ◽  
Vol 68 (7) ◽  
pp. 3496-3501 ◽  
Author(s):  
Eun-Sun Shin ◽  
Mi-Jeong Yang ◽  
Kyung Hwa Jung ◽  
Eun-Ju Kwon ◽  
Jae Sung Jung ◽  
...  
Keyword(s):  
Clostridium Thermocellum ◽  
Catalytic Domain ◽  
Binding Capacity ◽  
Residual Activity ◽  
Peptide Sequence ◽  
Optimum Temperature ◽  
Xylanase Gene ◽  
Signal Peptide Sequence ◽  
C Terminus ◽  
The One

ABSTRACT A xylanase gene, xynX, of Clostridium thermocellum had one thermostabilizing domain (TSD) between the signal peptide sequence and the catalytic domain (CD). The TSD of a truncated xylanase gene, xynX′TSD-CD, was transpositioned from the N terminus to the C terminus of the CD by overlapping PCRs, and a modified product, xynX′CD-TSD, was constructed. XynX′TSD-CD had a higher optimum temperature (70°C versus 65°C) and was more thermostable (residual activity of 68% versus 46% after a 20-min preincubation at 70°C) than the one without the TSD, XynX′CD. However, the domain-transpositioned enzyme, XynX′CD-TSD, showed a lower optimum temperature (30°C) and thermostability (20%) than XynX′CD. Both XynX′TSD-CD and XynX′CD-TSD showed significantly higher binding capacity toward xylan than XynX′CD, and the domain transposition did not cause any change in the binding ability. XynX′TSD-CD and XynX′CD-TSD also showed considerable binding to lichenan but not to carboxymethyl cellulose and laminarin. XynX′TSD-CD and XynX′CD-TSD had higher activities for insoluble xylan than XynX′CD, while XynX′CD was more active against soluble xylan than XynX′TSD-CD and XynX′CD-TSD. These results indicate that the TSD of XynX has dual functions, xylan binding and thermostabilization, and the domain should also be classified as a xylan-binding domain (XBD). The binding capacity of the XBD was not affected by domain transpositioning within the gene.

scholarly journals The eosinophil-specific cell surface antigen, EOS47, is a chicken homologue of the oncofetal antigen melanotransferrin

Blood ◽  
1996 ◽  
Vol 87 (4) ◽  
pp. 1343-1352 ◽  
Author(s):  
KM McNagny ◽  
F Rossi ◽  
G Smith ◽  
T Graf
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Peripheral Blood ◽  
Tertiary Structure ◽  
Surface Glycoprotein ◽  
Peptide Sequence ◽  
Sequence Information ◽  
Specific Cell ◽  
Specific Marker ◽  
Cell Surface Glycoprotein

The EOS47 antigen is a 100-kD cell surface glycoprotein selectively expressed by avian retrovirus-transformed eosinophils and their precursors. We have purified the EOS47 protein to homogeneity and used peptide sequence information to clone EOS47-encoding cDNAs. The open reading frames from these cDNAs predict a 738 amino acid protein with homology to human melanotransferrin, a membrane-found, transferrin-like protein that is expressed at high levels by a subset of melanomas, tumor cell lines, fetal intestine, and liver, but not by most normal adult tissues. The predicted protein sequence of EOS47 displays a 61% sequence identity with melanotransferrin and conservation of all 28 cysteine residues, indicating a similar tertiary structure. The finding that EOS47 lacks several of the iron-coordinating amino acids present in all transferrins suggests that it may be impaired in its ability to bind iron. In nonhematopoietic tissues, EOS47 is expressed at high levels by epithelial brush borders of small intestine and kidney and at lower levels by cells lining the sinusoids of the liver. Within hematopoietic tissues, EOS47 is restricted to a subpopulation of cells (1% to 5%) in bone marrow and early spleen and fluorescence-activated cell sorting of EOS47+ cells leads to a dramatic ( > 30-fold) enrichment of peroxidase+ eosinophils. In contrast, peripheral blood eosinophils are EOS47-, suggesting that the antigen is expressed by newly formed eosinophils and that expression ceases shortly before these cells emigrate from the bone marrow into the peripheral blood. Our results show that melanotransferrin is a stage-specific marker of eosinophils and should be useful for their isolation and further characterization.

scholarly journals Uroplakins Ia and Ib, two major differentiation products of bladder epithelium, belong to a family of four transmembrane domain (4TM) proteins.

1994 ◽  
Vol 125 (1) ◽  
pp. 171-182 ◽  
Author(s):  
J Yu ◽  
J H Lin ◽  
X R Wu ◽  
T T Sun
Keyword(s):  
Transmembrane Domain ◽  
Structure And Function ◽  
Amino Acid Sequences ◽  
Transmembrane Domains ◽  
Lung Epithelial Cells ◽  
Apical Surface ◽  
Bladder Epithelium ◽  
Differentiation Markers ◽  
Protein Particles ◽  
And Function

The mammalian bladder epithelium elaborates, as a terminal differentiation product, a specialized plasma membrane called asymmetric unit membrane (AUM) which is believed to play a role in strengthening and stabilizing the urothelial apical surface through its interactions with an underlying cytoskeleton. Previous studies indicate that the outer leaflet of AUM is composed of crystalline patches of 12-nm protein particles, and that bovine AUMs contain three major proteins: the 27- to 28-kD uroplakin I, the 15-kD uroplakin II and the 47-kD uroplakin III. As a step towards elucidating the AUM structure and function, we have cloned the cDNAs of bovine uroplakin I (UPI). Our results established the existence of two isoforms of bovine uroplakin I: a 27-kD uroplakin Ia and a 28-kD uroplakin Ib. These two glycoproteins are closely related with 39% identity in their amino acid sequences. Hydropathy plot revealed that both have four potential transmembrane domains (TMDs) with connecting loops of similar length. Proteolytic digestion of UPIa inserted in vitro into microsomal vesicles suggested that its two main hydrophilic loops are exposed to the luminal space, possibly involved in interacting with the luminal domains of other uroplakins to form the 12-nm protein particles. The larger loop connecting TMD3 and TMD4 of both UPIa and UPIb contains six highly conserved cysteine residues; at least one centrally located cysteine doublet in UPIa is involved in forming intramolecular disulfide bridges. The sequences of UPIa and UPIb (the latter is almost identical to a hypothetical, TGF beta-inducible, TI-1 protein of mink lung epithelial cells) are homologous to members of a recently described family all possessing four transmembrane domains (the "4TM family"); members of this family include many important leukocyte differentiation markers such as CD9, CD37, CD53, and CD63. The tissue-specific and differentiation-dependent expression as well as the naturally occurring crystalline state of uroplakin I molecules make them uniquely suitable, as prototype members of the 4TM family, for studying the structure and function of these integral membrane proteins.

scholarly journals Characterisation and functional analysis of canine TLR5

2020 ◽  
Vol 26 (6) ◽  
pp. 451-458
Author(s):  
Aihua Zhu ◽  
Lingling Wei ◽  
Sujuan Hu ◽  
Cheng Yang ◽  
Caifa Chen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Salmonella Enteritidis ◽  
Transmembrane Domain ◽  
Immunofluorescence Assay ◽  
Peptide Sequence ◽  
Signal Peptide Sequence ◽  
Reading Frame ◽  
Serovar Typhimurium ◽  
Receptor Domain

In this study, we characterised the single exon TLR5 gene of the Chinese rural dog. Sequence analysis revealed a 2577 nucleotide-long open reading frame of canine TLR5, encoding an 858 amino acid-long protein. The putative amino acid sequence of canine TLR5 consisted of a signal peptide sequence, 15 LRR domains, a LRR C-terminal domain, a transmembrane domain and an intracellular Toll-IL-1 receptor domain. The amino acid sequence of the canine TLR5 protein shared 95.4% identity with vulpine, 72.2% with feline and 64.7% with human TLR5. Plasmids expressing canine TLR5 and NF-κB-luciferase were constructed and transfected into HEK293T cells. Expression was confirmed by indirect immunofluorescence assay. These HEK293T cells transfected with the canine TLR5- and NF-κB-luciferase plasmids significantly responded to flagellin from Salmonella enteritidis serovar Typhimurium, indicating that it is a functional TLR5 homolog. In response to stimulation with Salmonella enteritidis, the level of TLR5 mRNA significantly increased over the control in PBMCs at 4 h. The levels of IL-8, IL-6 and IL-1β also increased after exposure. The highest levels of TLR5, IL-8 and IL-1β expression were detected at 8, 4 and 12 h after stimulation, respectively. These results imply that the expression of canine TLR5 may participate in the immune response against bacterial pathogens.

scholarly journals Reliable Reference Genes for Gene Expression Assessment in Tendon-Derived Cells under Inflammatory and Pro-Fibrotic/Healing Stimuli

Cells ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 1188 ◽  
Author(s):  
Enrico Ragni ◽  
Carlotta Perucca Orfei ◽  
Annie C. Bowles ◽  
Laura de Girolamo ◽  
Diego Correa
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Collagen Type I ◽  
Surface Glycoprotein ◽  
Type I ◽  
Cell Surface Glycoprotein ◽  
Tissue Healing ◽  
Tendon Cells ◽  
The Stability ◽  
Main Component

Tendon cells (TCs) are important for homeostatic maintenance in the healthy tendon and to promote tissue healing after injury. Further, resident and rare populations of tendon stem/progenitor cells, located at various sites within the tendon, contribute to tendon recovery by differentiating into repairing TCs. Gene expression analysis, through quantitative reverse-transcription polymerase chain reaction (qRT-PCR), constitutes a useful tool to study cellular responses, including the transition from initial inflammation to healing processes. A critical step required for data normalization is the choice of reliable reference genes (RGs), a process highly underestimated in tendon biology. In this study, the suitability of five commonly used RGs (ACTB, B2M, GAPDH, HPRT1, and RPLP0) was evaluated using TCs samples cultured in both standard and progenitor-enriching conditions, as well as under either inflammatory (IFNγ + TNFα) or pro-fibrotic/healing (CTGF) stimulation. The stability of the candidate RGs was computationally determined using NormFinder, geNorm, BestKeeper, and DeltaCt applets. Overall, ACTB resulted as the most stable RG on the basis of the integration of each gene weight, whereas B2M and RPLP0 performed poorly. To further validate ACTB’s optimal performance, we evaluated the expression of ICAM1, coding for an immune-related cell surface glycoprotein, and COL1A1, encoding collagen type I that is the main component of the tendon extracellular matrix (ECM), both known to be modulated by inflammation. The expression of both genes was heavily affected by the RGs used. Consequently, when analyzing gene expression in tendon-derived cells subjected to various stimulatory protocols, the use of a suitable RG should be considered carefully. On the basis of our results, ACTB can be reliably used when analyzing different TC types exposed to pathological conditions.

scholarly journals Synthesis and extracellular deposition of fibronectin in chondrocyte cultures. Response to the removal of extracellular cartilage matrix.

1978 ◽  
Vol 79 (2) ◽  
pp. 342-355 ◽  
Author(s):  
W Dessau ◽  
J Sasse ◽  
R Timpl ◽  
F Jilek ◽  
K von der Mark
Keyword(s):  
Type I Collagen ◽  
Sodium Dodecyl ◽  
Type Ii Collagen ◽  
Collagen Fibers ◽  
Cartilage Matrix ◽  
Surface Glycoprotein ◽  
Type I ◽  
Double Labeling ◽  
Cell Surface Glycoprotein

Fibronectin, the major cell surface glycoprotein of fibroblasts, is absent from differentiated cartilage matrix and chondrocytes in situ. However, dissociation of embryonic chick sternal cartilage with collagenase and trypsin, followed by inoculation in vitro reinitiates fibronectin synthesis by chondrocytes. Immunofluorescence microscopy with antibodies prepared against plasma fibronectin (cold insoluble globulin [CIG]) reveals fibronectin associated with the chondrocyte surface. Synthesis and secretion of fibronectin into the medium are shown by anabolic labeling with [35S]methionine or [3H]glycine, and identification of the secreted proteins by immunoprecipitation and sodium dodecyl sulfate (SDS)-disc gel electrophoresis. When chondrocytes are plated onto tissue culture dishes, the pattern of surface-associated fibronectin changes from a patchy into a strandlike appearance. Where epithelioid clones of polygonal chondrocytes develop, only short strands of fibronectin appear preferentially at cellular interfaces. This pattern is observed as long as cells continue to produce type II collagen that fails to precipitate as extracellular collagen fibers for some time in culture. Using the immunofluorescence double-labeling technique, we demonstrate that fibroblasts as well as chondrocytes which synthesize type I collagen and deposit this collagen as extracellular fibers show a different pattern of extracellular fibronectin that codistributes in large parts with collagen fibers. Where chondrocytes begin to accumulate extracellular cartilage matrix, fibronectin strands disappear. From these observations, we conclude (a) that chondrocytes synthesize fibronectin only in the absence of extracellular cartilage matrix, and (b) that fibronectin forms only short intercellular "stitches" in the absence of extracellular collagen fibers in vitro.

TIM-3, a Leukemia Stem Cell Marker, Plays a Role In Leukemic Transformation Through Autocrine Stimulatory Signaling By Its Ligand, Galectin-9

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4196-4196
Author(s):  
Yoshikane Kikushige ◽  
Junichiro Yuda ◽  
Takahiro Shima ◽  
Toshihiro Miyamoto ◽  
Koichi Akashi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myeloid Leukemia ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Surface Glycoprotein ◽  
Leukemic Cells ◽  
Stem Cell Marker ◽  
Cell Surface Glycoprotein ◽  
Hematopoietic Stem

Abstract Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. We have reported that the T-cell immunoglobulin mucin-3 (TIM-3) is expressed on LSCs in most types of AML but not on normal hematopoietic stem cells (HSCs) (Kikushige et al, Cell Stem Cell, 2010). We extended the analysis of TIM-3 expression into various types of human hematological malignancies, and found that human TIM-3 is expressed in the vast majority of CD34+CD38- LSCs of human myeloid malignancies including chronic myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndromes (MDS). Although CD34+CD38- normal bone marrow stem cells do not express TIM-3, TIM-3 is expressed in the CD34+CD38- population in MDS, and is further up-regulated with progression into leukemia. The average percentages of TIM-3+ cells in the CD34+CD38- population was 7.8% in RCMD (n=10), 19.2% in RAEB-1 (n=10), 84.0% in RAEB-2 (n=10) and 92.2% in overt AML (n=10). The close association of TIM-3 expression with transformation into AML led us to hypothesize that TIM-3 itself has a function in AML stem cell development. TIM-3 is a type 1 cell-surface glycoprotein and has a structure that includes an N-terminal immunoglobulin variable domain followed by a mucin domain, a transmembrane domain and a cytoplasmic tail. Tyrosine residues are clustered in the cytoplasmic tail, suggesting that TIM-3 can induce signal transduction in TIM-3+ AML cells. To understand the function of TIM-3, we investigated the interaction between TIM-3 and its ligand galectin-9 in AML LSCs. We found that AML patients showed significantly higher serum galectin-9 concentration than healthy individuals (healthy controls: 18.3+4.3 pg/ml, AML patients: 139.1+33.4 pg/ml, P<0.05). Unexpectedly, we found that leukemic cells expressed a high level of galectin-9 protein, as compared to other hematopoietic cells including T cells, B cells and monocytes. Using KASUMI-3 (TIM-3+ AML cell line) and primary AML samples, we confirmed that AML cells could secrete galectin-9 after TLR stimulation in vitro. Furthermore, microarray analysis demonstrated that TIM-3 stimulation by the physiological concentration of galectin-9 induced significant gene expression changes toward pro-survival axis including up-regulation of MCL-1, the important survival factor for HSCs and LSCs. These results collectively suggest that AML cells can produce and secrete galectin-9, and galectin-9 can bind and stimulate TIM-3-expressing AML cells including LSCs in an autocrine manner to support their survival or leukemia progression. Disclosures: Miyamoto: Kyushu University Hospital: Employment.

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