Structural analysis and expression of human desmoglein: a cadherin-like component of the desmosome

1991 ◽  
Vol 99 (4) ◽  
pp. 809-821
Author(s):  
L.A. Nilles ◽  
D.A. Parry ◽  
E.E. Powers ◽  
B.D. Angst ◽  
R.M. Wagner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Adhesion ◽  
Structural Analysis ◽  
Calcium Binding ◽  
Human Keratinocytes ◽  
Cell Junctions ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Extracellular Domains

Desmosomes are adhesive cell junctions found in great abundance in tissues that experience mechanical stress. The transmembrane desmosomal glycoproteins have been proposed to play a role in cell adhesion; desmoglein I (DGI) is a major member of this class of desmosomal molecules. However, evidence supporting a role for DGI in cell adhesion or in the plaque is lacking. In order to begin to understand DGI function we have identified human cDNA clones encoding the entire mature polypeptide of 1000 amino acids. Our data suggest that like the bovine DGI molecule human DGI is highly related to the calcium-dependent class of cell adhesion molecules known as cadherins. Four related extracellular domains located in the amino-terminal domain of the molecule contain putative calcium binding sites originally identified in the cadherins. The highest degree of similarity between human N-cadherin and human DGI, and likewise between bovine DGI and human DGI, is greatest in the most amino-terminal extracellular domain. This suggests a conserved functional role for the extracellular domains, perhaps in calcium-mediated cell adhesion. The cytoplasmic portion of the molecule contains a cadherin-like region and, like bovine DGI, a carboxy-terminal tail that is not present in the cadherins, comprising three additional domains. One of these contains a novel repeating motif of 29 +/− 1 residues, first identified in bovine DGI. Each of the highly homologous repeating units is likely to consist of two beta-strands and two turns with special characteristics. Five amino acids that are identical in bovine and human DGI lie in the second of the two predicted beta-strands, and intriguingly contain putative target sites for protein kinase C. On the basis of structural analysis, a model predicting the disposition of human DGI domains in the desmosome is proposed. Northern analysis suggests that unlike bovine epidermis, which expresses a single mRNA of reported size approximately 7.6 kb, human foreskin and cultured keratinocytes display a complex pattern with bands of approximately 7.2, 4.0 and 3.0 kb. Each of these cross-hybridizing mRNAs is coordinately expressed in normal human keratinocytes in response to long-term culture and increased calcium.

The sequence and tissue expression of ovine renin

1992 ◽  
Vol 8 (1) ◽  
pp. 3-11 ◽  
Author(s):  
G. P. Aldred ◽  
P. Fu ◽  
R. J. Crawford ◽  
R. T. Fernley
Keyword(s):  
Amino Acids ◽  
Cdna Probe ◽  
Tissue Expression ◽  
Nucleotide Sequences ◽  
Northern Analysis ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Untranslated Sequence ◽  
Gene Coding ◽  
Renin Mrna

ABSTRACT The primary structure of the sheep renin precursor has been determined from its cDNA sequence. A library of cDNA clones was constructed from adrenalectomized sheep kidney poly(A)+ RNA and screened for sheep renin sequences with a cloned mouse renin cDNA probe. Of the 300 000 clones generated, 24 were hybridization positive and the nucleotide sequences of two of the longest clones were determined. These clones coded for the mature sheep renin protein and the 3′-untranslated sequence but did not extend to the amino-terminal region of preprorenin. Clones corresponding to the 5′ region of renin mRNA were generated by the polymerase chain reaction and their nucleotide sequences determined. The sheep renin precursor consists of 400 amino acids with a putative leader sequence of 14 amino acids and a putative 45 or 53 amino acid prosegment. The mature sheep renin protein has a 73% sequence identity with human renin. Northern analysis demonstrated the presence of renin mRNA in the kidney but not in other tissues in the sheep. While sodium depletion of sheep caused a rise in renin mRNA in the kidney, adrenalectomy also led to a large increase in renal renin mRNA. Southern analysis of genomic DNA suggests that there is only one gene coding for renin in the sheep.

scholarly journals Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily.

1991 ◽  
Vol 112 (5) ◽  
pp. 1017-1029 ◽  
Author(s):  
M P Burgoon ◽  
M Grumet ◽  
V Mauro ◽  
G M Edelman ◽  
B A Cunningham
Keyword(s):  
Cell Adhesion ◽  
Adhesion Molecule ◽  
Cell Adhesion Molecule ◽  
Single Gene ◽  
Structural Features ◽  
Cdna Clones ◽  
Glia Cell ◽  
Type Iii ◽  
Amino Terminal ◽  
Extracellular Region

The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.

scholarly journals Fimbrin is a homologue of the cytoplasmic phosphoprotein plastin and has domains homologous with calmodulin and actin gelation proteins.

1990 ◽  
Vol 111 (3) ◽  
pp. 1069-1079 ◽  
Author(s):  
M V de Arruda ◽  
S Watson ◽  
C S Lin ◽  
J Leavitt ◽  
P Matsudaira
Keyword(s):  
Calcium Binding ◽  
Actin Binding ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Actin Binding Domain ◽  
Solid Tissues ◽  
Complete Protein ◽  
Carboxy Terminal ◽  
Amino Terminal Domain

Fimbrin is an actin-bundling protein found in intestinal microvilli, hair cell stereocilia, and fibroblast filopodia. The complete protein sequence (630 residues) of chicken intestine fimbrin has been determined from two full-length cDNA clones. The sequence encodes a small amino-terminal domain (115 residues) that is homologous with two calcium-binding sites of calmodulin and a large carboxy-terminal domain (500 residues) consisting of a fourfold-repeated 125-residue sequence. This repeat is homologous with the actin-binding domain of alpha-actinin and the amino-terminal domains of dystrophin, actin-gelation protein, and beta-spectrin. The presence of this duplicated domain in fimbrin links actin bundling proteins and gelation proteins into a common family of actin cross-linking proteins. Fimbrin is also homologous in sequence with human L-plastin and T-plastin. L-plastin is found in only normal or transformed leukocytes where it becomes phosphorylated in response to IL 1 or phorbol myristate acetate. T-plastin is found in cells of solid tissues where it does not become phosphorylated. Neoplastic cells derived from solid tissues express both isoforms. The differences in expression, sequence, and phosphorylation suggest possible functional differences between fimbrin isoforms.

scholarly journals The primary structure of NG2, a novel membrane-spanning proteoglycan.

1991 ◽  
Vol 114 (2) ◽  
pp. 359-371 ◽  
Author(s):  
A Nishiyama ◽  
K J Dahlin ◽  
J T Prince ◽  
S R Johnstone ◽  
W B Stallcup
Keyword(s):  
Amino Acids ◽  
Chondroitin Sulfate ◽  
Primary Structure ◽  
Disulfide Bonds ◽  
Transmembrane Domain ◽  
Core Protein ◽  
Extracellular Domain ◽  
Cdna Clones ◽  
Amino Terminal ◽  
Extracellular Region

The complete primary structure of the core protein of rat NG2, a large, chondroitin sulfate proteoglycan expressed on O2A progenitor cells, has been determined from cDNA clones. These cDNAs hybridize to an mRNA species of 8.9 kbp from rat neural cell lines. The total contiguous cDNA spans 8,071 nucleotides and contains an open reading frame for 2,325 amino acids. The predicted protein is an integral membrane protein with a large extracellular domain (2,224 amino acids), a single transmembrane domain (25 amino acids), and a short cytoplasmic tail (76 amino acids). Based on the deduced amino acid sequence and immunochemical analysis of proteolytic fragments of NG2, the extracellular region can be divided into three domains: an amino terminal cysteine-containing domain which is stabilized by intrachain disulfide bonds, a serine-glycine-containing domain to which chondroitin sulfate chains are attached, and another cysteine-containing domain. Four internal repeats, each consisting of 200 amino acids, are found in the extracellular domain of NG2. These repeats contain a short sequence that resembles the putative Ca(++)-binding region of the cadherins. The sequence of NG2 does not show significant homology with any other known proteins, suggesting that NG2 is a novel species of integral membrane proteoglycan.

scholarly journals Structure of a new nervous system glycoprotein, Nr-CAM, and its relationship to subgroups of neural cell adhesion molecules.

1991 ◽  
Vol 113 (6) ◽  
pp. 1399-1412 ◽  
Author(s):  
M Grumet ◽  
V Mauro ◽  
M P Burgoon ◽  
G M Edelman ◽  
B A Cunningham
Keyword(s):  
Nervous System ◽  
Cell Adhesion ◽  
Transmembrane Domain ◽  
Neuronal Cell ◽  
Immunofluorescent Staining ◽  
Cdna Clones ◽  
Distribution Analysis ◽  
Type Iii ◽  
Amino Terminal ◽  
Lentil Lectin

We have identified and characterized a new glycoprotein in the chicken nervous system using immunological and molecular biological methods and we have examined its tissue distribution. Analysis revealed that this protein is very similar in structure to the chicken neuron-glia cell adhesion molecule, Ng-CAM, and to mouse L1. cDNA clones encompassing the entire coding sequence of this Ng-CAM related molecule, called Nr-CAM, have been isolated and sequenced. A glycoprotein containing one major component of Mr 145,000 on SDS-PAGE was purified from brain by lentil lectin affinity chromatography and FPLC, and its amino-terminal sequence was identical to that predicted from the Nr-CAM cDNA. The complete cDNA sequence encodes six Ig-like domains, five fibronectin type III repeats, a predicted transmembrane domain, and a short cytoplasmic domain. On Northern blots, nucleic acid probes for Nr-CAM recognized one major RNA species of approximately 7 kb and much lesser amounts of larger RNAs. Most of the same probes hybridized to single bands on genomic Southern blots, suggesting that Nr-CAM is encoded by a single gene that may be alternatively processed to yield several mRNAs. In support of this notion, two Nr-CAM cDNA clones had a 57-bp sequence located between the second and third Ig-like domains that was not found in two other Nr-CAM cDNA clones, and two other clones were isolated that lacked the 279-bp segment encoding the fifth fibronectin-like type III repeat. Antibodies against the purified protein and synthetic peptides in Nr-CAM both recognized a predominant Mr 145,000 species and a much less prevalent species of Mr 170,000 in neural tissues. Levels of Nr-CAM expression increased in the brain until approximately embryonic day (E) 12, followed by slightly lower levels of expression at E18 and after hatching. Immunofluorescent staining with anti-Nr-CAM antibodies showed that most neurons in the retina were positive at E7 and the pattern of expression became restricted to several layers on neuronal cell bodies and fibers during development. Anti-Nr-CAM antibodies labeled specifically cell surfaces on neurons in culture. Although the structure of Nr-CAM resembles that of chicken Ng-CAM and mouse L1, the identity with each of these neural CAMs does not exceed 40%. The differences indicate that Nr-CAM is distinct from Ng-CAM and L1, but there are sufficient similarities to suggest that all of these molecules are members of a subgroup of neural CAMs in the N-CAM superfamily.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen.

1987 ◽  
Vol 7 (9) ◽  
pp. 3221-3230 ◽  
Author(s):  
N Beauchemin ◽  
S Benchimol ◽  
D Cournoyer ◽  
A Fuks ◽  
C P Stanners
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Carcinoembryonic Antigen ◽  
Full Length ◽  
Cdna Clones ◽  
Base Pairs ◽  
Amino Terminal ◽  
Isolation And Characterization

Carcinoembryonic antigen (CEA) expression is perhaps the most prevalent of phenotypic changes observed in human cancer cells. The molecular genetic basis of this phenomenon, however, is completely unknown. Twenty-seven CEA cDNA clones were isolated from a human colon adenocarcinoma cell line. Most of these clones are full length and consist of a number (usually three) of surprisingly similar long (534 base pairs) repeats between a 5' end of 520 base pairs and a 3' end with three different termination points. The predicted translation product of these clones consists of a processed signal sequence of 34 amino acids, an amino-terminal sequence of 107 amino acids, which includes the known terminal amino acid sequence of CEA, three repeated domains of 178 amino acids each, and a membrane-anchoring domain of 27 amino acids, giving a total of 702 amino acids and a molecular weight of 72,813 for the mature protein. The repeated domains have conserved features, including the first 67 amino acids at their N termini and the presence of four cysteine residues. Comparisons with the amino acid sequences of other proteins reveals homology of the repeats with various members of the immunoglobulin supergene family, particularly the human T-cell receptor gamma chain. CEA cDNA clones in the SP-65 vector were shown to produce transcripts in vitro which could be translated in vitro to yield a protein of molecular weight 73,000 which in turn could be precipitated with CEA-specific antibodies. CEA cDNA clones were also inserted into an animal cell expression vector and introduced by transfection into mammalian cell lines. These transfectants produced a CEA-immunoprecipitable glycoprotein which could be visualized by immunofluorescence on the cell surface.

scholarly journals Structural Characteristics of Prothrombin

1977 ◽  
Author(s):  
Daniel A. Walz ◽  
Walter H. Seegers
Keyword(s):  
Amino Acids ◽  
Calcium Binding ◽  
Structural Characteristics ◽  
Amino Acid Sequences ◽  
National Institutes Of Health ◽  
Sequence Information ◽  
Binding Region ◽  
Amino Terminal ◽  
A Chain ◽  
Β Sheet

The amino acid sequences of human and bovine prothrombin fragments 1 and 2 have been completed. In addition, partial sequence information has been obtained on the respective fragments from chicken prothrombin. Fragment 1 from human (155 amino acids), bovine (156 amino acids) and chicken (approximately 161 amino acids) contains the vitamin K-dependent calcium binding region (residues 1-32) and is a highly conserved region of the fragment. Fragment 2 from human (118 amino acids), bovine (118 amino acids) and chicken (113 amino acids) is apparently undergoing a more rapid rate of nucleotide substitution than is fragment 1. The A-chain of the enzyme from human (36+13 amino acids), bovine (49 amino acids) and chicken (49 amino acids) have a highly variable amino-terminal portion (residues 1-13) and a conserved portion (residues 14-49). Calculations for fragment 1 indicate the presence of 3α-helical regions (23.2%), 9β-sheet regions (34.2%) and 10β-turns (25.8%). The calcium binding region is an α-helical region of the molecule. Fragment 2 has 5α-helical regions (38.1%), 4β-sheet regions (19.5%) and 9 β-turns (30.5%). The differing rates of substitution found for fragments 1 and 2 are having no apparent effect on the calculated α-helical or β-sheet regions within prothrombin and may account for the limited, conserved activation bonds available within the prothrombin molecule. (Supported in part by a grant from the National Institutes of Health, HL-03424-19 and the McGregor Foundation of Detroit).

scholarly journals A role for the epidermal growth factor-like domain of P-selectin in ligand recognition and cell adhesion

1994 ◽  
Vol 124 (4) ◽  
pp. 609-618 ◽  
Author(s):  
GS Kansas ◽  
KB Saunders ◽  
K Ley ◽  
A Zakrzewicz ◽  
RM Gibson ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Ligand Recognition ◽  
Detectable Effect ◽  
High Endothelial Venules ◽  
Direct Role ◽  
Parent Molecule ◽  
Lectin Domain ◽  
Amino Terminal ◽  
Extracellular Domains ◽  
Extracellular Region

The selectin family of adhesion molecules mediates the initial interactions of leukocytes with endothelium. The extracellular region of each selectin contains an amino-terminal C-type lectin domain, followed by an EGF-like domain and multiple short consensus repeat units (SCR). Previous studies have indirectly suggested a role for each of the extracellular domains of the selectins in cell adhesion. In this study, a panel of chimeric selectins created by exchange of domains between L- and P-selectin was used to directly examine the role of the extracellular domains in cell adhesion. Exchange of only the lectin domains between L- and P-selectin conferred the adhesive and ligand recognition functions of the lectin domain of the parent molecule. However, chimeric selectins which contained both the lectin domain of L-selectin and the EGF-like domain of P-selectin exhibited dual ligand-binding specificity. These chimeric proteins supported adhesion both to myeloid cells and to high endothelial venules (HEV) of lymph nodes and mesenteric venules in vivo. Exchange of the SCR domains had no detectable effect on receptor function or specificity. Thus, the EGF-like domain of P-selectin may play a direct role in ligand recognition and leukocyte adhesion mediated by P-selectin, with the lectin plus EGF-like domains collectively forming a functional ligand recognition unit.

scholarly journals The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

1986 ◽  
Vol 103 (5) ◽  
pp. 1635-1648 ◽  
Author(s):  
J Lawler ◽  
R O Hynes
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Sites ◽  
Calcium Binding ◽  
Cdna Clones ◽  
Coding Region ◽  
Calcium Binding Sites ◽  
Mediate Cell ◽  
Type 3

Thrombospondin is one of a class of adhesive glycoproteins that mediate cell-to-cell and cell-to-matrix interactions. We have used two monoclonal antibodies to isolate cDNA clones of thrombospondin from a human endothelial cell cDNA library and have determined the complete nucleotide sequence of the coding region. Three regions of known amino acid sequence of human platelet thrombospondin confirm that the clones are authentic. Three types of repeating amino acid sequence are present in thrombospondin. The first is 57 amino acids long and shows homology with circumsporozoite protein from Plasmodium falciparum. The second is 50-60 amino acids long and shows homology with epidermal growth factor precursor. The third occurs as a continuous eightfold repeat of a 38-residue sequence; structural homology with parvalbumin and calmodulin indicates that these repeats constitute the multiple calcium-binding sites of thrombospondin. The amino acid sequence arg-gly-asp-ala is included in the last type 3 repeat. This sequence is probably the site for the association of thrombospondin with cells. In addition, localized homologies with procollagen, fibronectin, and von Willebrand factor are present in one region of the thrombospondin molecule.

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