The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain.

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.

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Bernard-Soulier Syndrome Caused by a Dinucleotide Deletion and Reading Frameshift in the Region Encoding the Glycoprotein Ibα Transmembrane Domain

Blood ◽

10.1182/blood.v90.7.2634.2634_2634_2643 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2634-2643 ◽

Cited By ~ 1

Author(s):

Vahid Afshar-Kharghan ◽

José A. López

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Transmembrane Domain ◽

Silent Mutation ◽

Cell Surface Expression ◽

Unaffected Individual ◽

Exon 2 ◽

The Third ◽

Bernard Soulier Syndrome

We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ibα was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ibα degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ibα, GP Ibβ, and GP IX genes. The patient was homozygous for a specific GP Ibα allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ibα gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T → C) in the second nucleotide of exon 2, which is in the 5′ untranslated region of the GP Ibα transcript, and a silent mutation in the third base of the codon for Arg342 (A → G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ibα amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ibα cDNA, alone and in combination with the 5′ mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ibα expression on the cell surface. Expression was not decreased further by addition of the 5′ mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ibβ, as demonstrated by their coimmunoprecipitation with a GP Ibβ antibody.

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Hyaluronan-binding region of aggrecan from pig laryngeal cartilage. Amino acid sequence, analysis of N-linked oligosaccharides and location of the keratan sulphate

Biochemical Journal ◽

10.1042/bj2860761 ◽

1992 ◽

Vol 286 (3) ◽

pp. 761-769 ◽

Cited By ~ 31

Author(s):

F P Barry ◽

J U Gaw ◽

C N Young ◽

P J Neame

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Signal Peptidase ◽

Amino Acid Residues ◽

Terminal Sequence ◽

Binding Region ◽

Keratan Sulphate ◽

Laryngeal Cartilage ◽

Hyaluronan Binding

The hyaluronan-binding region (HABR) was prepared from pig laryngeal cartilage aggrecan and the amino acid sequence was determined. The HABR had two N-termini: one N-terminal sequence was Val-Glu-Val-Ser-Glu-Pro (367 amino acids in total), and a second N-terminal sequence (Ala-Ile-Ser-Val-Glu-Val; 370 amino acids in total) was found to arise due to alternate cleavage by the signal peptidase. The N-linked oligosaccharides were analysed by examining their reactivity with a series of lectins. It was found that the N-linked oligosaccharide on loop A was of the mannose type, while that on loop B was of the complex type. No reactivity was detected between the N-linked oligosaccharide on loop B' and any of the lectins. The location of keratan sulphate (KS) in the HABR was determined by Edman degradation of the immobilized KS-containing peptide. The released amino acid derivatives were collected and tested for the presence of epitope to antibody 5-D-4. On the basis of 5-D-4 reactivity and sequencing yields, the KS chains are attached to threonine residues 352 and 357. There is no KS at threonine-355. This site is not in fact in G1, but about 16 amino acid residues into the interglobular domain. Comparison of the structure of the KS chain from the HABR and from the KS domain of pig laryngeal cartilage aggrecan was made by separation on polyacrylamide gels of the oligosaccharides arising from digestion with keratanase. Comparison of the oligosaccharide maps suggests that the KS chains from both parts of the aggrecan molecule have the same structure.

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Molecular cloning and expression of the cDNA for alpha 3 subunit of human alpha 3 beta 1 (VLA-3), an integrin receptor for fibronectin, laminin, and collagen.

The Journal of Cell Biology ◽

10.1083/jcb.115.1.257 ◽

1991 ◽

Vol 115 (1) ◽

pp. 257-266 ◽

Cited By ~ 88

Author(s):

Y Takada ◽

E Murphy ◽

P Pil ◽

C Chen ◽

M H Ginsberg ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

General Structure ◽

Chinese Hamster ◽

Cloning And Expression ◽

Cdna Libraries ◽

Cdna Clones ◽

Alpha Subunits

alpha 3 beta 1 (VLA-3), a member of the integrin family of cell adhesion receptors, may function as a receptor for fibronectin, laminin, and collagen. A partial cDNA clone (2.4 kb) for the human alpha 3 subunit was selected from an endothelial cell lambda gt11 cDNA library by specific antibody screening. Several overlapping cDNA clones were subsequently obtained, of a total length of 4.6 kb from various cDNA libraries. The reconstructed alpha 3 cDNA was expressed on the surface of chinese hamster ovary cells as detected by an alpha 3-specific mAb after transfection, suggesting that the cDNA is authentic. Within this sequence was an open reading frame, encoding for 1,051 amino acids, including a signal peptide of 32 residues, a long extracellular domain (959 residues), a transmembrane domain (28 residues), and a short cytoplasmic segment (32 residues). Overall, the alpha 3 amino acid sequence was 25-37% similar to the other integrin alpha subunits that are cleaved, with most similarity to the alpha 6 sequence (37%), and less similarity to those alpha subunits that have I domains (15-20%, excluding the I domain sequence itself). Features most like those in other alpha subunits are (a) the positions of 18/19 cysteine residues, (b) three potential metal binding domains of the general structure DX(D/N)X(D/N)GXXD, and (c) the predicted transmembrane domain. The mass of alpha 3 calculated from its amino acid sequence is 113,505. The human alpha 3 sequence was 89% identical to hamster galactoprotein b3, and 70% similar to the chicken CSAT antigen band 2 protein partial sequence, suggesting that these two polypeptides are homologues of human alpha 3.

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THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V

10.1055/s-0038-1643887 ◽

1987 ◽

Author(s):

Richard J Jenny ◽

Debra D Pittman ◽

John J Toole ◽

Ronald W Kriz ◽

Randal J Kaufman ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Factor Viii ◽

Untranslated Region ◽

Human Factor ◽

Leader Peptide ◽

Cdna Clones ◽

Factor V ◽

Human Factor Viii

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)

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Bernard-Soulier Syndrome Caused by a Dinucleotide Deletion and Reading Frameshift in the Region Encoding the Glycoprotein Ibα Transmembrane Domain

Blood ◽

10.1182/blood.v90.7.2634 ◽

1997 ◽

Vol 90 (7) ◽

pp. 2634-2643 ◽

Cited By ~ 28

Author(s):

Vahid Afshar-Kharghan ◽

José A. López

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Transmembrane Domain ◽

Silent Mutation ◽

Cell Surface Expression ◽

Unaffected Individual ◽

Exon 2 ◽

The Third ◽

Bernard Soulier Syndrome

Abstract We investigated the molecular genetic and biosynthetic basis of Bernard-Soulier syndrome in a severely affected white woman. Flow cytometric analysis showed a severe deficiency of glycoprotein (GP) Ib, GP IX, and GP V on the surface of her platelets. Similarly, GP Ibα was undetectable by immunoblot analysis of platelet lysates. Surprisingly, a large quantity of a 70-kD protein (which probably represents a GP Ibα degradation product) was found in the patient's plasma in much greater quantities than in the plasma of an unaffected individual. To analyze the molecular lesion responsible for the disorder, we amplified and sequenced gene segments corresponding to the entire coding regions of the GP Ibα, GP Ibβ, and GP IX genes. The patient was homozygous for a specific GP Ibα allele that contained two tandem VNTR repeats in the region encoding the macroglycopeptide (C variant) and three differences from the published GP Ibα gene sequence. Two mutations were unlikely to be involved in the disorder: the substitution of a single base (T → C) in the second nucleotide of exon 2, which is in the 5′ untranslated region of the GP Ibα transcript, and a silent mutation in the third base of the codon for Arg342 (A → G) that does not change the amino acid sequence. The third mutation was a deletion of the last two bases of the codon for Tyr492 (TAT). This mutation causes a frameshift that alters the GP Ibα amino acid sequence, beginning within its transmembrane region. The mutant polypeptide contains 81 novel amino acids and is 38 amino acids shorter than its wild-type counterpart. The new sequence changes the hydrophobic nature of the transmembrane domain and greatly decreases the net positive charge of what had been the cytoplasmic domain. The deletion mutation was introduced into the GP Ibα cDNA, alone and in combination with the 5′ mutation, and expressed in Chinese hamster ovary (CHO) cells. The deletion alone severely reduced GP Ibα expression on the cell surface. Expression was not decreased further by addition of the 5′ mutation, confirming that the deletion was the cause of the Bernard-Soulier phenotype. Stable cell lines expressing the mutant polypeptide secreted large amounts of the polypeptide into the medium, suggesting that the mutant anchors poorly in the plasma membrane. Nevertheless, a fraction of the mutant was able to associate with GP Ibβ, as demonstrated by their coimmunoprecipitation with a GP Ibβ antibody.

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Isolation and sequence of a cDNA for human pro-(cathepsin L)

Biochemical Journal ◽

10.1042/bj2530303 ◽

1988 ◽

Vol 253 (1) ◽

pp. 303-306 ◽

Cited By ~ 76

Author(s):

S Gal ◽

M M Gottesman

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Amino Acid Sequence Identity ◽

Cathepsin L ◽

Acid Proteinase ◽

Cdna Clones ◽

Terminal Sequence ◽

Human Form ◽

Cross Hybridization ◽

Human Cathepsin

The major excreted protein (MEP) of malignantly transformed mouse fibroblasts is the precursor to an acid proteinase with enzymic specificity similar to that of human cathepsin L. By cross-hybridization with a mouse MEP sequence, cDNA clones of the human form of MEP in an SV40 expression vector were isolated. A 1.6 kb cDNA showed 70% deduced amino acid sequence identity with mouse MEP. The deduced amino acid sequence of the cloned human MEP was the same, except for two amino acids, as the N-terminal sequence of mature human cathepsin L, thereby establishing that human MEP is human pro-(cathepsin L). Use of this human pro-(cathepsin L) cDNA clone allowed the detection of a 1.6-1.8 kb pro-(cathepsin L) mRNA in human cells which was not detected with a mouse pro-(cathepsin L) probe.

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ISOLATION AND CHARACTERIZATION OF cDNA CLONES FOR THE PLATELET MEMBRANE GLYCOPROTEINS IIb and IIIa

10.1055/s-0038-1643961 ◽

1987 ◽

Author(s):

A E Butler-Zimrin ◽

J S Bennett ◽

M Poncz ◽

E Schwartz ◽

S Surrey ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Transmembrane Domain ◽

Platelet Membrane ◽

Membrane Receptors ◽

Cdna Clones ◽

Single Chain ◽

Platelet Protein ◽

Isolation And Characterization ◽

Terminal Amino

The platelet membrane GPIIb/GPIIIa complex on activated platelets contains receptors for fibrinogen, von Willebrand factor, and fibronectin. GPIIb and GPIlia also appear to be members of a family of membrane receptors involved in cell-cell and cell-matrix interactions. To study the structure of GPIIb and GPIIIa, we have constructed an expression library in the vector lambda gtll using mRNA from the HEL cell line and screened it with polyclonal antibody against each platelet protein. HEL cells constitutively express proteins similar to platelet GPIIb and GPIIIa. A 3.2kb GPIIb cDNA clone was identified that encodes for all 1008 amino acids of GPIIb including the known N-terminal amino acids of the α Cand βsubunits. This confirms that GPIIb is synthesized as a single chain polypeptide that is cleaved into two disulfide-linked subunits posttranslation. Analysis of the amino acid sequence revealed a major C-terminal transmembrane domain in the βsubunit, two potential transmembrane domains near the N-terminus of the αsubunit, and four possible N-linked glycosylation sites. Approximately 30% amino acid identity was found between GPIIb and the available amino acid sequences for the larger chains of the fibronectin and vitronectin receptors. Initial sequence analysis of a 3.8kb cDNA for GPIIIa included the known N-terminal amino acids of the platelet protein. Northern blot analysis was performed using HEL cell total RNA. The GPIIb cDNA hybridized to a 4.1kb mRNA while the GPIIIa cDNA hybridized to a 5.8kb mRNA. This indicates that the two cDNAs do not cross-hybridize and suggests that GPIIb and GPIIIa are encoded by separate genes. The availability of these cDNA for GPIIb and GPIIIa will facilitate study of the structure and function of the proteins and will aid in clarifying their relationship to other adhesive protein receptors.

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Amino acid sequence of penicillopepsin. IV. Myxobacter AL-1 protease II and Staphylococcus aureus protease fragments and homology with pig pepsin and chymosin

Canadian Journal of Biochemistry ◽

10.1139/o76-128 ◽

1976 ◽

Vol 54 (10) ◽

pp. 902-914 ◽

Cited By ~ 19

Author(s):

Anne Cunningham ◽

Hsin-Min Wang ◽

Stephen R. Jones ◽

Alexander Kurosky ◽

Leticia Rao ◽

...

Keyword(s):

Amino Acids ◽

Staphylococcus Aureus ◽

Amino Acid ◽

Amino Acid Sequence ◽

Sequence Data ◽

Amino Acid Residues ◽

Terminal Sequence ◽

Fungal Enzyme ◽

Fungal Protease ◽

And Staphylococcus Aureus

The digest of penicillopepsin (EC 3.4.23.7) with protease II from Myxobacter AL-1 gave five fragments which were separated on a Biogel P-100 column in 70% formic acid. The fragments were from 16 to 125 amino acids long. Two fragments were also isolated from a digest with a protease from Staphylococcus aureus. The analysis of these fragments by automatic sequencer gave a number of overlaps of the chymotryptic and thermolytic peptides. The available amino acid sequence data for penicillopepsin described in this paper and the accompanying papers (Kurosky, A. &Hofmann, T.: Can. J. Biochem. 54, 872 (1976); Rao, L. &Hofmann, T.: Can. J. Biochem. 54, 885 (1976); Harris, C. I., Rao, L., Shutsa, P., Kurosky, A. &Hofmann, T.: Can. J. Biochem. 54, 895 (1976)) have been combined and yield 15 fragments which range in lengths from 3 to 112 amino acid residues. These unique fragments account for virtually all the amino acids of the fungal protease. Four of the fragments with a total of 194 residues (about 60% of the molecule) have been aligned with corresponding sections of pig pepsin (EC 3.4.23.1) and with part of the N-terminal sequence available for calf chymosin (EC 3.4.23.4). In the alignments about 37% of the residues in the fungal enzyme are identical with at least one of the mammalian enzymes. An additional 20% are chemically similar. These results, together with previously reported active-site directed modifications, show conclusively that penicillopepsin is an evolutionary homologue of the mammalian acid proteases.

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Molecular cloning of cDNA for porcine prolactin precursor

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0040135 ◽

1990 ◽

Vol 4 (2) ◽

pp. 135-142 ◽

Cited By ~ 3

Author(s):

Y. Kato ◽

T. Hirai ◽

T. Kato

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Signal Sequence ◽

Cdna Clones ◽

Amino Acid Residues ◽

Ancestral Gene ◽

Wide Range ◽

Hydrophilic Residues ◽

Ovine Prolactin

ABSTRACT Porcine prolactin cDNA clones were screened using antiserum against ovine prolactin from a cDNA library of porcine anterior pituitary, and their nucleotide sequences were determined by the chain-termination method. The nucleotide sequence of the 5′ untranslated region and part of the signal peptide region were determined by direct RNA sequencing with reverse transcriptase. The composite sequence of 957 nucleotides showed a signal sequence of 30 amino acids and a further 199 amino acids corresponding to the mature prolactin molecule. The predicted sequence confirmed the amino acid sequence determined previously by direct protein analysis, except for one amide form at residue 122 (Gln instead of the reported Glu). Northern blot analysis showed that the length of the porcine prolactin mRNA was about 1·1 kb. The porcine prolactin amino acid sequence showed 81, 80, 64, 62, 80 and 31% homology with human, bovine, rat, mouse, chick and salmon forms respectively. The identical amino acid residues showed marked clustering in four domains, two of which are highly conserved throughout a wide range of species. The hydropathy and secondary structure of porcine prolactin were analysed and compared with those of porcine GH, which shares the same ancestral gene. The two highly conserved regions of both hormones showed similar hydrophilicity, and the predicted secondary structures indicated that these regions in each hormone form different structures with differences in extension of the hydrophilic residues outside the molecule.

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Functional Mapping of an Oligomeric Autotransporter Adhesin of Aggregatibacter actinomycetemcomitans

Journal of Bacteriology ◽

10.1128/jb.01709-07 ◽

2008 ◽

Vol 190 (9) ◽

pp. 3098-3109 ◽

Cited By ~ 22

Author(s):

Chunxiao Yu ◽

Teresa Ruiz ◽

Christopher Lenox ◽

Keith P. Mintz

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Matrix Protein ◽

Aggregatibacter Actinomycetemcomitans ◽

Binding Activity ◽

Extracellular Matrix Protein ◽

Deletion Mutants ◽

Mature Protein ◽

Collagen Binding ◽

Transmission Electron

ABSTRACT Extracellular matrix protein adhesin A (EmaA) is a 202-kDa nonfimbrial adhesin, which mediates the adhesion of the oral pathogen Aggregatibacter actinomycetemcomitans to collagen. EmaA oligomers form surface antenna-like protrusions consisting of a long helical rod with an ellipsoidal ending. The functional analysis of in-frame emaA deletion mutants has located the collagen binding activity to the amino terminus of the protein corresponding to amino acids 70 to 386. The level of collagen binding of this deletion mutant was comparable to the emaA mutant strain. Transmission electron microscopy studies indicate that the first 330 amino acids of the mature protein form the ellipsoidal ending of the EmaA protrusions, where the activity resides. Amino acid substitution analysis within this sequence has identified a critical amino acid, which is essential for the formation of the ellipsoidal ending and for collagen binding activity.

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