Identification of Novel Placentally Expressed Aspartic Proteinase in Humans
Abstract
:1. Introduction
2. Results
2.1. Identification of cDNA Sequence Originating from Term Placental Transcriptome
2.2. Identification of cDNA Sequence Originating from Term Placental Transcriptome
2.3. Identification of the hPAG-L/pep Sequence within the Human Genome
2.4. Identification of Placental hPAG-L/pep Protein
2.5. Identification of Cellular hPAG-L/pep Localization
3. Discussion
3.1. Identification of hPAG-L Transcript
3.2. Identification of hPAG-L Exonic-Intronic Structure
3.3. Identification of hPAG-L Proteins
3.4. Identification of Cellular hPAG-L Localization
4. Materials and Methods
4.1. Ethics Statement and Collection of Samples
4.2. Total RNA Extraction
4.3. High Throughput mRNA and Bioinformatics
4.4. Capillary Sequencing
4.5. Genomic Identification of the hAP/PAG-L Sequence
4.6. Identification of the Exon-Intron Organization of the hPAG-L
4.7. Cellular Placental Protein Extraction
4.8. Western Blotting
4.9. Heterologous Double Fluorescent Immunohistochemistry (htdF-IHC)
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
PAG | Pregnancy-associated glycoprotein family |
PAG-L | PAG-like |
hPAG-L | Human PAG-L |
Pep | Pepsinogen |
RNA-seq | RNA sequencing |
aa | Amino acids |
D | Asparagine (Asp) |
cDNA | Complementary DNA |
gDNA | Genomic DNA |
bPAG | Bovine PAG |
pPAG | Porcine PAG |
CfPAG-L | Beaver PAG-L |
EbPAG | European bison PAG |
LpPAG | Lama pacos PAG |
CdPAG | Camelus dromedarius PAG |
CfPAG | Camelus ferrus PAG |
AaPAG-L | Alces alces PAG |
RIA | Radioimmunological test |
ELISA | Immunoenzymatic test |
RT-PCR | Reverse transcriptase PCR |
dNTP | Deoxynucleotide |
MWCO | Molecular weight cut-off |
SDS-PAGE | Denaturing polyacrylamide electrophoresis |
CBB | Coomassie Brilliant Blue |
NBT | Nitro blue tetrazolium |
BCIP | 5-Bromo-4-chloro-3-indolyl-phosphate |
ht | Heterologous (cross-species) |
anti-pPAG-P | Polyclonals raised against various porcine antigens |
anti-Rec pPAG2 | Polyclonals raised against recombinant pPAG2 antigen |
htdF-IHC | Heterologous double fluorescent immunohistochemistry |
A488 | Alexa 488 fluorophore |
PI | Propidium iodide |
HQ | High quality |
CDS | Coding sequence |
UTR | Untranslated region |
QC | Query Cover |
SP | Signal peptide |
ID | Sequence identity |
CV | Chorionic villi |
VC | Villous core |
FC | Fetal capillaries |
IS | Intervillous space |
PS | Placental septa |
SB | Stratum basale |
NC | Negative control |
dpc | Day post coitum |
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Gene Name a | SP Sequence (aa) b | Identity (%) | Positive aa (%) |
---|---|---|---|
hPAG-L/pep | MKWLLLLGLVALSEC | this study | this study |
hPepsinogen A | ............... | 100 | 100 |
bPAG2 | ....V.......... | 93.3 | 100 |
pPAG1 | ....VI......... | 86.7 | 100 |
mPepsinogen F | ....WV......... | 86.7 | 93.3 |
fPAG | ....WV......... | 86.7 | 93.3 |
pPAG2 | ....VI.......D. | 80 | 100 |
ePAG | ...FGV....T.... | 73.3 | 80 |
CfPAG-L | ...IVVA.LC.P.L.A | 37.5 | 62.5 |
hCathepsin E | ..T....L..L.ELGEAQG | 60 | 60 |
hPepsinogen C | ...MVVV-..C.QLLEA | 40 | 66.7 |
hNapsin A | QPL....P.LNVEPSGA | 33.3 | 46.7 |
hRenin | PR.G..--.LLWGS.TFG | 33.3 | 46.7 |
hCathepsin D | .QPSS..P.ALCLLAAPASA | 26.7 | 33.3 |
Gene Name a | Blocking Peptide Sequence (aa) b | Identity (%) | Positive aa (%) |
---|---|---|---|
hPAG-L/pep | IMYKVPLIRKKSLRRTLSERGLLKDFLKKHNLNPARKYFPQWEAPTL | this study | this study |
hPepsinogen A | ............................................... | 100 | 100 |
hPepsinogen C | AVV....KKF..I.E.MK.K...GE..RT.KYD..W..R.GDL | 46.5 | 65.1 |
fPAG | -LVTI..T.V..M.EN.R.KDR.....EN.PY.L.Y.FVD | 43.6 | 59 |
pPAG2 | -LVMI..TKV..V.ES.R.K....N...E.PY.MIQNL | 43.2 | 67.6 |
CfPAG-L | AISRI..RKA..V.Q..K.K...EE...T.KYD..Q..LANNFGDF | 41.3 | 65.2 |
pPAG1 | -LVII..TKV..I.EN.R.KD..LN...E.PY.MIQ.F | 40.5 | 64.9 |
ePAG | -LVTI..VKI....EN.R.KDM..EY.E.YPFRL | 36.4 | 66.7 |
mPepsinogen F | -LV.I..MKI..M.EN.R.SQV...Y.E.YPRSR.HVLLE.RRN. | 36.4 | 59.1 |
bPAG2 | .VIL-..KKM.T..E..R.KN..NN..EEQAYRLSKNDS | 33.3 | 56.4 |
Gene Name a | NH2-Domain b | Identity (%) | COOH-Domain b | Identity (%) |
---|---|---|---|---|
hPAG-L/pep. | VVFDTGSSNLWV | this study | AIVDTGTSLLTG | this study |
hPepsinogen A | ............. | 100 | ............ | 100 |
hCathepsin D | ............ | 100 | .........MV. | 83.3 |
hPepsinogen C | .L.......... | 91.7 | ...........V | 91.7 |
hCathepsin E | .I.......... | 91.7 | .........I.. | 91.7 |
CfPAG-L | .L.......... | 91.7 | G..........V | 83.3 |
TrNothepsin | ........D... | 91.7 | .........IA. | 83.3 |
hNapsin A | .A.......... | 91.7 | ..L......I.. | 83.3 |
pPAG2, 4, 6, 10 | ........D... | 91.7 | ........MLH. | 75 |
oPAG2 | ........D... | 91.7 | .L.......IH. | 75 |
bPAG2 | .......A.... | 91.7 | .LL.....MIY. | 58.3 |
hRenin A | .........V.. | 91.7 | .L....A.YIS. | 58.3 |
mPepsinogen F | ..L.....V... | 83.3 | G.M......... | 83.3 |
fPAG | .I......D... | 83.3 | ..I.......I. | 83.3 |
ePAG | .I.....AD... | 75 | ..........L. | 91.7 |
zPAG | .I.....AD... | 75 | ..........L. | 91.7 |
pPAG1, 3, 5 | .I...A..D... | 75 | ..L.S.SAF.L. | 50 |
Sequence Length (bp) | |||||
---|---|---|---|---|---|
Gene Segment | hPAG-L/pep | bPAG1 | bPAG2 | pPAG2 | CfPAG-L |
Exon 1 | 56 | 53 | 53 | 53 | 59 |
Intron A | 486 | 1100 | 1300 | 1093 | 1937 |
Exon 2 | 163 | 151 | 151 | 166 | 160 |
Intron B | 2233 | 1000 | 1000 | 1324 | 385 |
Exon 3 | 118 | 118 | 118 | 118 | 118 |
Intron C | 869 | 100 | 100 | 90 | 917 |
Exon 4 | 119 | 119 | 119 | 119 | 119 |
Intron D | 675 | 1200 | 1200 | 1124 | 451 |
Exon 5 | 200 | 194 | 194 | 200 | 200 |
Intron E | 1351 | 900 | 1100 | 927 | 1138 |
Exon 6 | 117 | 117 | 117 | 117 | 123 |
Intron F | 1131 | 1900 | 1700 | 1455 | 288 |
Exon 7 | 145 | 142 | 142 | 136 | 148 |
Intron G | 102 | 100 | 100 | 85 | 681 |
Exon 8 | 99 | 99 | 99 | 99 | 99 |
Intron H | 1119 | 1700 | 1700 | 292 | 603 |
Exon 9 | 150 | 150 | 150 | 156 | 147 |
Total length | 9133 | 9143 | 9343 | 8031 | 7573 |
Donor Splice Sites | Acceptor Splice Sites | ||||||||
---|---|---|---|---|---|---|---|---|---|
Exon | 5′→3′ | Phase | Intron | 5′→3′ | Intron | 5′→3′ | Phase | Exon | 5′→3′ |
1 | TCATGTACAA | 0 | A | GTGAGTCCGG | A | CAAACCACAG | 2 | 2 | GGTCCCCCTC |
2 | CTACCTGGAT | 0 | B | GTGAGTGTGC | B | GCCTGGACAG | 0 | 3 | ATGGAGTACT |
3 | CTTGCCTGCA | 1 | C | GTAAGTGCCC | C | GTCCTTGCAG | 1 | 4 | CCAACCACAA |
4 | CACTGTCCAG | 0 | D | GTGGGCACCT | D | CCCCACCCAG | 0 | 5 | GTTGGAGGCA |
5 | ACCTCAGCGC | 2 | E | GTAAGTTGAG | E | CTTTCCACAG | 2 | 6 | CGATGACCAG |
6 | CCGTGGACAG | 2 | F | GTGAGACTGC | F | TTGCCCTCAG | 2 | 7 | CATCACCATG |
7 | AGATGGCGAC | 0 | G | GTGAGTCCAG | G | CTCTTTCCAG | 0 | 8 | ATGGTGGTCA |
8 | CATCCTGCAG | 0 | H | GTGAGGAGGC | H | TTTTCTCCAG | 0 | 9 | AGCGAGGGGA |
Pairwise Identity (%) | |||
---|---|---|---|
hPAG-L/pep | bPAG1 | pPAG2 | CfPAG-L |
Exon 1 | 78.6 | 75.5 | 63.3 |
Intron A | 49.8 | 51.1 | 50.3 |
Exon 2 | 52.1 | 58.8 | 59.6 |
Intron B | 51.7 | 50.7 | 52.7 |
Exon 3 | 69.5 | 74.6 | 71.2 |
Intron C | 55.6 | 54.5 | 25.4 |
Exon 4 | 59.7 | 58.4 | 64.7 |
Intron D | 52.5 | 52.2 | 52.7 |
Exon 5 | 58.5 | 65.3 | 65.5 |
Intron E | 51.3 | 53.3 | 50.9 |
Exon 6 | 61.9 | 69.7 | 56.9 |
Intron F | 51.2 | 51.0 | 50.3 |
Exon 7 | 62.0 | 63.3 | 64.7 |
Intron G | 58.5 | 54.2 | 54.7 |
Exon 8 | 67.7 | 66.7 | 70.3 |
Intron H | 52.9 | 52.3 | 51.8 |
Exon 9 | 58.7 | 61.4 | 61.5 |
Primers Name | Sequence (5′–3′) | Position (bp) a | Amplicon Length (bp) a | |
---|---|---|---|---|
1 | MMstart | AGTTGGGACCCGGGAAGA | 1–18 | 1363 |
MMutrR | TCCACAAAACCTGTTTCAGTG | 1343–1364 | ||
2 | MM2s | TCATCAGAAAGAAGTCCTTGAG | 85–106 | 496 |
MM5as | TAGGCCAGSCCCAKGATGCCATC | 558–580 | ||
3 | MM3s | GCTCCTCCAACCTGTGGGT | 307–325 | 560 |
MM7as | CAGAGAGGTGCCKGTGTCMACAA | 844–866 | ||
4 | MM5s | GATGGCATCMTGGGSCTGGCCTA | 558–580 | 564 |
MM9as | GAAGACATCWCCMAGGATCCAA | 1100–1121 | ||
5 | MM7s | TTGTKGACACMGGCACCTCTCTG | 844–866 | 520 |
MMutrR | TCCACAAAACCTGTTTCAGTG | 1343–1364 |
Primers Name | Sequence (5′–3′) | Position (bp) a | Amplicon Length (bp) a | |
---|---|---|---|---|
1 | MMstart | AGTTGGGACCCGGGAAGA | 1–18 | 725 |
MM2as | ATCCAGGTAGTTCTCCAGGG | 706–725 | ||
2 | MM2s | TCATCAGAAAGAAGTCCTTGAG | 571–592 | 1789 |
MMintronBr | ATTCTCCTGCCTCAACCTCCCAA | 2337–2359 | ||
3 | MMintronB | CTCCGCATAGCCTGATCCCTT | 1180–1200 | 1180 |
MMintronBr | ATTCTCCTGCCTCAACCTCCCAA | 2337–2359 | ||
4 | MMintronB3 | CCTCCTGCAGATATTGTATGTCC | 1429–1451 | 1616 |
MM3as | ACCCACAGGTTGGAGGAGCC | 3025–3044 | ||
5 | MMintronB2 | TGTGAGGAATGAAGGAAAAGATGG | 2840–2863 | 1533 |
MMintronDr | GGTGCTGCATGTCGGGAGAA | 4353–4372 | ||
6 | MMintronC | GCTGTAGAATAGCCCACCAGG | 3381–3401 | 992 |
MMintronDr | GGTGCTGCATGTCGGGAGAA | 4353–4372 | ||
7 | MMintronC | GCTGTAGAATAGCCCACCAGG | 3381–3401 | 1663 |
MMintronEr | AAGACCCTCTCCATCGCACCCA | 5022–5043 | ||
8 | MMintronD(N) | AGTCCTGCATGAGATGAACCA | 4636–4656 | 1284 |
MMintronEr3 | CTTAAGGACTTGAGGGTGGAGGTC | 5896–5919 | ||
9 | MMintronE3 | GCACAACTCAAATGTCATCAGCCA | 5178–5201 | 742 |
MMintronEr3 | CTTAAGGACTTGAGGGTGGAGGTC | 5896–5919 | ||
10 | MMintronE3 | GCACAACTCAAATGTCATCAGCCA | 5178–5201 | 1325 |
MMintronFr2 | CTGGGGGGATTCTGGAAAGCTGA | 6480–6502 | ||
14 | MM6sens | AGTGGCAGCGTGGTGATCTTTG | 6301–6322 | 1311 |
MM7as | CAGAGAGGTGCCKGTGTCMACAA | 7589–7611 | ||
15 | MMintronF2 | TCAGCTTTCCAGAATCCCCCCAG | 6480–6502 | 1132 |
MM7as | CAGAGAGGTGCCKGTGTCMACAA | 7589–7611 | ||
16 | MMintronF | TGGATGGGTGGGGAAGAAATGT | 7438–7459 | 1650 |
MM9as | GAAGACATCWCCMAGGATCCAA | 9066–9087 | ||
17 | MM8s | GACATCGTCTTCACCATCAAT | 7822–7842 | 1266 |
MM9as | GAAGACATCWCCMAGGATCCAA | 9066–9087 | ||
18 | MMintronF | TGGATGGGTGGGGAAGAAATGT | 7438–7459 | 405 |
MM8as | ATTGATGGTGAAGACGATGTC | 7822–7842 | ||
19 | MM992 | GAGCTGCATCAGTGGCTTCC | 9012–9031 | 318 |
MMutrR | TCCACAAAACCTGTTTCAGTG | 9309–9329 |
© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Majewska, M.; Lipka, A.; Panasiewicz, G.; Gowkielewicz, M.; Jozwik, M.; Majewski, M.K.; Szafranska, B. Identification of Novel Placentally Expressed Aspartic Proteinase in Humans. Int. J. Mol. Sci. 2017, 18, 1227. https://doi.org/10.3390/ijms18061227
Majewska M, Lipka A, Panasiewicz G, Gowkielewicz M, Jozwik M, Majewski MK, Szafranska B. Identification of Novel Placentally Expressed Aspartic Proteinase in Humans. International Journal of Molecular Sciences. 2017; 18(6):1227. https://doi.org/10.3390/ijms18061227
Chicago/Turabian StyleMajewska, Marta, Aleksandra Lipka, Grzegorz Panasiewicz, Marek Gowkielewicz, Marcin Jozwik, Mariusz Krzysztof Majewski, and Bozena Szafranska. 2017. "Identification of Novel Placentally Expressed Aspartic Proteinase in Humans" International Journal of Molecular Sciences 18, no. 6: 1227. https://doi.org/10.3390/ijms18061227