Mammalian expression of full-length bovine aggrecan and link protein: Formation of recombinant proteoglycan aggregates and analysis of proteolytic cleavage by ADAMTS-4 and MMP-13

Abstract Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP’d with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

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Epitope Mapping of a Monoclonal Antibody against VWF Which Diminishes the Proteolytic Cleavage of VWF by ADAMTS13 Under Flow.

Blood ◽

10.1182/blood.v114.22.2133.2133 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2133-2133

Author(s):

Jingyu Zhang ◽

Zhenni Ma ◽

Ningzheng Dong ◽

Jian Su ◽

Anyou Wang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Proteolytic Activity ◽

Recombinant Proteins ◽

Expression System ◽

Guanidine Hydrochloride ◽

Proteolytic Cleavage ◽

Terminal Region ◽

Full Length ◽

Western Blots ◽

A2 Domain

Abstract Abstract 2133 Poster Board II-110 Introduction: In our former study, we have found that SZ-34, a monoclonal antibody to Von Willebrand factor (VWF), can inhibit the proteolysis of VWF by ADAMTS13 under shear stress. But the precise epitope of this antibody (SZ-34) on VWF is not clear for it is generated by immunizing mouse with native full-length VWF purified from pooled human normal plasmas. Thus, the objective of this study is to map the epitope of SZ-34 and to explore the effect of VWF structrue on the proteolytic activity by ADAMTS13. Materials and Methods: Firstly we constructed and expressed a series of recombinant proteins of different domains or polypeptide fragments of human VWF in prokaryotic cell expression system, including A1A2A3, D′D3, A1, A2, A3, A1A2, A2A3 and five sub-fragments of A2 domain. Then native VWF and these recombinant proteins or polypeptide fragments were subjected to polyacrylamide gel electrophoresis (PAGE) and analyzed by Western blots with SZ-34. Results: Different recombinant proteins of VWF were successfully expressed and purified. Results of Western blot showed that SZ-34 could bind specifically some recombinant proteins, such as full-length VWF, A1A2A3, A2 and GST-D1459D1596 in which the last was a fusion protein of a sub-fragment of A2 domain with GST. But SZ-34 couldn't bind to others, including A1, A3, D′D3, GST-D1459E1554, GST-E1554D1596, GST-D1596R1668 (VWF73) and GST- E1554R1668. In addition, the reacting activity of SZ-34 with native VWF was significantly stronger than with unfolded VWF, such as heat-treated or 1.5M guanidine hydrochloride-treated VWF. Conclusions: The epitope of SZ-34 is located within N-terminal region fore-VWF73 inside VWF-A2 domain. Besides, SZ-34 maybe is a conformation-specific monoclonal antibody. Combining with our former findings that SZ-34 inhibits the proteolytic cleavage of VWF by ADAMTS13, we can conclude that N-terminal region fore-VWF73 inside VWF-A2 domain also regulates the proteolytic activity of VWF by ADAMTS13, although VWF73 is considered as the minimal substrate for ADAMTS13. Disclosures: No relevant conflicts of interest to declare.

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Firefly luciferase gene: structure and expression in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.2.725 ◽

1987 ◽

Vol 7 (2) ◽

pp. 725-737 ◽

Cited By ~ 1789

Author(s):

J R de Wet ◽

K V Wood ◽

M DeLuca ◽

D R Helinski ◽

S Subramani

Keyword(s):

Mammalian Cells ◽

Firefly Luciferase ◽

Full Length ◽

Expression Vectors ◽

Luciferase Expression ◽

Luciferase Gene ◽

S1 Nuclease ◽

Reading Frame ◽

Mammalian Expression ◽

Firefly Luciferase Gene

The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression.

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C/EBPα Regulates Generation of C/EBPβ Isoforms through Activation of Specific Proteolytic Cleavage

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.1695 ◽

1999 ◽

Vol 19 (3) ◽

pp. 1695-1704 ◽

Cited By ~ 78

Author(s):

Alana L. Welm ◽

Nikolai A. Timchenko ◽

Gretchen J. Darlington

Keyword(s):

Translational Control ◽

Cultured Cells ◽

Proteolytic Cleavage ◽

Full Length ◽

In Vitro Translation ◽

Translation System ◽

Cleavage Activity ◽

Intronless Genes ◽

Transcriptional Activator Protein ◽

Alternative Translation

ABSTRACT C/EBPα and C/EBPβ are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPβ has been reported to produce four isoforms: full-length 38-kDa C/EBPβ, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. In this report, we investigated the mechanisms by which C/EBPβ isoforms are generated in the liver and in cultured cells. Using an in vitro translation system, we found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism—specific proteolytic cleavage of full-length C/EBPβ. Studies of mice in which the C/EBPα gene had been deleted (C/EBPα−/−) showed that the regulation of C/EBPβ proteolysis is dependent on C/EBPα. The induction of C/EBPα in cultured cells leads to induced cleavage of C/EBPβ to generate the LIP isoform. We characterized the cleavage activity in mouse liver extracts and found that the proteolytic cleavage activity is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBPα−/− animals. The lack of cleavage activity in the livers of C/EBPα−/− mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration showed that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. We propose that there are two mechanisms by which C/EBPβ isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPα-dependent, specific proteolytic cleavage of full-length C/EBPβ. The latter mechanism implicates C/EBPα in the regulation of posttranslational generation of the dominant negative C/EBPβ isoform, LIP.

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The role of link-protein in the structure of cartilage proteoglycan aggregates

Biochemical Journal ◽

10.1042/bj1770237 ◽

1979 ◽

Vol 177 (1) ◽

pp. 237-247 ◽

Cited By ~ 262

Author(s):

T E Hardingham

Keyword(s):

Analytical Ultracentrifugation ◽

Free Form ◽

Link Protein ◽

Physiological Conditions ◽

Neutral Ph ◽

Laryngeal Cartilage ◽

Cartilage Proteoglycan ◽

Proteoglycan Aggregates

Proteoglycan fractions were prepared from pig laryngeal cartilage. The effect of link-protein on the properties of proteoglycan-hyaluronate aggregates was examined by viscometry and analytical ultracentrifugation. Aggregates containing link-protein were more stable than link-free aggregates at neutral pH, at temperatures up to 50 degrees C and in urea (up to 4.0M). Oligosaccharides of hyaluronate were able to displace proteoglycans from link-free aggregates, but not from the link-stabilized aggregates. Both types of aggregate were observed in the ultracentrifuge, but at the concentration investigated (less than 2 mg/ml) the link-free form was partially dissociated and the proportion aggregated varied with the pH and temperature and required more hyaluronate for saturation than did link-stabilized aggregate. The results showed that link-protein greatly strengthened the binding of proteoglycans to hyaluronate and suggest that under physiological conditions it ‘locks’ proteoglycans on to the hyaluronate chain.

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Demonstration of link protein in proteoglycan aggregates from human intervertebral disc

Biochemical Journal ◽

10.1042/bj2220085 ◽

1984 ◽

Vol 222 (1) ◽

pp. 85-92 ◽

Cited By ~ 18

Author(s):

A Tengblad ◽

R H Pearce ◽

B J Grimmer

Keyword(s):

Intervertebral Disc ◽

Density Gradient ◽

Gradient Centrifugation ◽

Buoyant Density ◽

Intervertebral Discs ◽

Human Articular Cartilage ◽

Link Protein ◽

Nasal Cartilage ◽

Proteoglycan Aggregates ◽

Cartilage Proteoglycans

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.

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Toward Understanding the Function of Amelogenin Using Transgenic Mice

Advances in Dental Research ◽

10.1177/08959374960100021501 ◽

1996 ◽

Vol 10 (2) ◽

pp. 208-214 ◽

Cited By ~ 1

Author(s):

K. Ibaraki-O'Connor ◽

K. Nakata ◽

M.F. Young

Keyword(s):

Southern Blotting ◽

Signal Sequence ◽

Transgenic Animals ◽

Full Length ◽

Pcr Analysis ◽

Coding Region ◽

Chain Reaction ◽

Mammalian Expression ◽

Ectopic Production ◽

Polymerase Chain

The purpose of this study was to establish transgenic mouse lines as a tool to investigate the function of amelogenin during mineralization by causing ectopic production of amelogenin and studying its effect. The mouse amelogenin (mAme) was cloned from a 16-day-old whole mouse embryo cDNA library and was determined to be "full-length" mouse amelogenin (with a complete coding region) by comparison with the mouse amelogenin reported previously by Snead et al. (1985) and Lau et al. (1992). The overexpression construct contained: (1) the rat osteocalcin (OC) promoter (1.8 kb); (2) the adenovirus splicing casettes, including introgenic (Int) sequence (0.3 kb); (3) the full-length mAme cDNA (0.8 kb); and (4) the polyadenylation signal sequence from the pSG5 mammalian expression vector. Both Southern blotting and polymerase chain-reaction (PCR) analyses were performed, by means of a specific probe and a pair of oligodeoxynucleotides to OclntmAme(A)+, respectively. The animals which showed transgene-positive in both analyses were further used to establish F1 animals. Heterozygocity was confirmed with F1 animals by PCR analysis of DNA from the F0 x FVB/N pups. Three independent transgenic F1 heterozygous lines (640t, 706t, and 708t) have now been established. The generation of F2 homozygous lines is under way. The heterozygous transgenic animals are currently being analyzed for alterations in the morphology and structure of various bone tissues.

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