|Lysyl oxidases: Cloning and characterization of the fourth and the fifth human lysyl oxidase isoenzymes, and the consequences of a targeted inactivation of the first described lysyl oxidase isoenzyme in mice|
Detailed descriptions of the material and methods are presented in the orginal papers I-III.
The BLASTN program (http://www.ncbi.nlm.nih.gov/BLAST/) was used to search all available databases for sequences that are similar to those of the coding regions of the human LOX (Hämäläinen et al. 1991), LOXL (Kenyon et al. 1993), and LOXL2 (Saito et al. 1997, Jourdan-Le Saux et al. 1999) cDNAs. Three high-throughput genomic sequences (AC005033, AC005041, AL139241) and one expressed sequence tag (AL751493) similar to LOXL2 sequence were found. Oligonucleotides for PCR were synthesized based on these sequences.
To identify cDNA clones for LOXL3, the PCR primer pairs HLO406F/HLO406R and HLO402F/HLO404R were used to generate 204 bp and 527 bp products from a human placenta γ gt11 cDNA library (Clontech). These PCR products were used to screen the same library, and two positive clones were obtained and characterized. To obtain the 5’end of the cDNA, the primers LO4cDNA1F and HLO406R were used for PCR with human placenta and fetus cDNA pools (Marathon Ready cDNA, Clontech). The PCR was performed using the Advantage 2 PCR Enzyme System kit (Clontech). Rapid amplification of the 5’ cDNA ends (5’RACE) was performed using the same cDNA pools as templates, and the Advantage 2 PCR Enzyme System kit with primer pairs AP1/HLO406R and AP1/LO4RNA1R. These PCR products were subcloned into the plasmid pUC18 (Amersham Pharmacia Biotech). All products were sequenced using standard methods.
To obtain cDNA clones for LOXL4, oligonucleotides HLO52F and HLO52R were used for PCR with a human kidney γ gt11 cDNA library (Clontech). The PCR product was then used for screening the same library, and a 1611 bp clone was identified. To obtain the 5’end of the cDNA, the oligonucleotide LO5E1AR was used with AP2 according to the Marthon-Ready cDNA User Manual (Clontech) and a 347 bp product was obtained from a human fetus cDNA pool.
The high throughput genomic sequences were compared with the cDNA sequences obtained using the exon prediction softwares GRAIL 1.3, FGENES, and Genie (http://dot.imgen.bcm.tmc.edu:9331/seq-search/gene-search.html). To verify the position of the first intron, PCR was performed using the placenta and fetus cDNA pools and Advantage 2 PCR Enzyme System kit (Clontech) with primer pairs HLO4prom4F/HLO406R and HLO4prom4F/LO4RNA1R, yielding products of the sizes 971 bp and 371 bp, respectively.
Human multi-tissue Northern blots I and II were used for analysis of the expression of LOXL3 and LOXL4 mRNA species. For LOXL3 mRNA, a 971 bp PCR product from the 5’end of the cDNA was used as a probe. This probe was obtained by PCR with primers HLO4prom4F and HLO406R. For LOXL4 mRNA, the corresponding full-length cDNA was used as a probe.
DNA sequencing was performed using an automated sequencer (ABI Prism 377, Applied Biosystems). DNASIS and PROSIS version 6.0 softwares (Pharmacia) were used to analyse the raw sequence data.
Recombinant expression of the LOXL3 and LOXL4 cDNAs was carried out using the mammalian expression vector pcDNA3.1/V5-HisA (Invitrogen). Expression constructs corresponding to the open reading frames of both cDNAs were generated by PCR with the human fetus cDNA pool as the template. These constructs contained, at their 3’ end, sequences encoding the V5 epitope and a His tag to be used for antibody staining of the recombinant protein. The plasmids were then transfected into human HT-1080 fibrosarcoma cells (American Type Culture Collection) using the Fugene6‘- transfection reagent (Boehringer Mannheim). The transfected HT-1080 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies, Inc.) containing 10% fetal bovine serum (FBS), 400 g/ml G-418 sulphate and 100 units/ml penicillin and streptomycin at 37°C in 5 % CO2. Lysates were prepared from confluent plates by removing the medium, rinsing the cell layers with PBS, and suspending the cells at 37°C in Hank’s balanced salt solution containing 0.05 % trypsin and 0.53 mM EDTA. After addition of an equal volume of 10 % FBS/DMEM, the cells were sedimented, and the cell pellet was resuspended and washed in PBS. The resulting pellet was suspended in PBS, and aliquots from the cells were lysed by adding a solution of 62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% β -mercaptoethanol, followed by heating at 100°C. For medium samples, confluent plates were cultured for 16 h, as above except that the medium contained no FBS. The samples for SDS/PAGE were concentrated using MicroSep‘ centrifugal 10 kDa concentrators (Filtron). All samples were analyzed by 8% SDS/PAGE and immunoblotting with a monoclonal anti-V5 antibody (Invitrogen).
LOXL3 transfected HT-1080 cells seeded on glass coverslips and grown to the desired density were fixed in pre-cooled methanol at -20°C and incubated in 2% BSA-PBS, pH 7.2, to reduce nonspecific staining. A monoclonal anti-V5 antibody was applied at an appropriate dilution, and the samples were incubated for 1 h at room temperature, followed by extensive washing with PBS. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibody was allowed to bind to the specimens for 1 h at room temperature. After extensive washing with PBS, the coverslips were mounted on microscope slides with Immu-Mount (Shandon) and viewed and photographed using a light microscope.
|Outlines of the present research||Generation and analysis of a mouse strain lacking the Lox gene (III)|