DNA Sequence-/.DNA Sequencing and Mapping, Vol. 1, pp. 275-277 Reprints available directly trom the publisher Photocopying permitted by license only
0 1991 Harwood Academic
Publishers GmbH Printed in the United Kingdom
SHORT COMMUNICATION
Differences in human laminin B2 sequences Mitochondrial DNA Downloaded from informahealthcare.com by University of Newcastle on 01/02/15 For personal use only.
CECILIA L.S. SANTOS, JORGE SABBAGA and RICARDO BRENTANI Ludwig Institute for Cancer Research Brazil
- Sao Paul0 Branch,
Rua Prof. Antonio Prudente, 109, 4 andar, 01509 Sao Paulo,
E M B L Data Library Accession No. X13939.
some clones were established in E . coli RY 1089 cells and gel1 electrophoretical analyses of the corresponding protein extracts showed that P-galactosidase fusion proteins were synthesized by the induced lysogens. The recombinant protein from one lysogen (pLB2) was electroeluted from preparative gels and used to raise antibodies in mice. Western blot analysis revealed that the anti-recombinant protein pLB2 antiserum recognizes laminin B chains both frorn EHS mouse and from human placenta (fig. 1A). Northern blot analysis (fig. 16) on RNA isolated from HT 1080 human fibrosarcoma cells and rnouse placenta showed two bands of 5,5 and 7,5 kb using
A cDNA clone encoding the B2 chain of laminin has been isolated from a human endothelial A g t l l cDNA library. The nucleotide and deduced amino acid sequences of the clone were determined and showed one amino acid substitution (Ser1519 instead of Asn) when compared to lung laminin 62 chain and one silent nucleotide change (C4200 instead of A) in relation to the human placenta laminin 62 chain. Other differences in the 3’untranslated region were also found.
Key words: basement membrane, extracellular matrix, dothelial cell, laminin, laminin 62 chain
en-
Laminin, a major component of basement membranes, is a large glycoprotein consisting of three distinct polypeptide chains (A= 400kD; B1 = 210kD; B2= 200kD) linked together by disulphide bonds in a cross-shaped structure (Engel et a/. 1981; Timpl and Martin 1982). This glycoprotein has many biological activities such as promoting cell adhesion, migration, mitogenesis, growth and differentiation. Some of these properties are the result of interactions with other components of basement membranes which associate with different domains of the laminin molecule (Martin and Timpl 1987). The isolation of basement membrane genes will provide an opportunity to study the molecular mechanisms of these complex processes. For this purpose two 3, g t l l cDNA (EC-1: 3’oligo dT- and EC-2: hexanucleotide random primed) libraries (a gift from Dr. Ginsburg, University of Michigan), constructed using mRNA from human umbilical vein endothelial cells (Ginsburg et a/., 1985) were immunoscreened with an affinity purified polyclonal rabbit antiserum directed against mouse laminin extracted from the Engelbreth-Holm-Swarm (EHS) tumor. Positive clones showed cDNA inserts ranging from 600 to 3000 bp. Southern blot experiments showed that the clones isolated from the EC-1 library cross-hybridize with those isolated from the EC-2 library. Lysogens of
kD
A
B 1
2
3
1
kb 2
400-
- 7.5
200-
- 5.5
Figure 1 , (A) lmmunoreaction of antibodies to recombinant protein pLB2 with laminin chains. Laminin preparations isolated from human placenta and EHS mouse tumor were analysed by SDS-PACE, transferred to nitrocellulose filter and visualized with anti recombinant pLB2 antiserum. Lane 1 : mouse laminin; lane 2: human placenta laminin; lane 3: pLB2 recombinant protein. Molecular markers are shown on the left; (B) Northern blot analysis. Total RNA (20ug) from mouse placenta (lane 1) and human HT 1080 fibrosarcoma cells (lane 2) were hybridized with nick translated pLB2 clone. Transcript sizes, given in kilobase pairs (kb) were estimated by comparison with the positions of 28s and 18s ribosomal RNA on the gel.
275
276
C . L. S. SANTOS ETAL.
A CTG GAG AAT GAA GCA AAT AAC ATA AAG ATG GAA GCT GAG AAT ZTG GAA CAA L E N E A N N I K M E A E N L E Q
37%
CTG ATT GAC CAG AAA TTA AAA GAT TAT GAG GAC CTC AGA GAA GAT ATG AGA GGG i I D Q K L K D Y E D L R E D M R G AAG K
1;AA
E
CTT GAA GTC AAG AAC CTT CTG GAG AAA GGC AAG ACT GAA CAG CAG ACC L E V K N L L E K G K T E Q Q T
30G3
1361
Mitochondrial DNA Downloaded from informahealthcare.com by University of Newcastle on 01/02/15 For personal use only.
GCA GAC CAA CTC CTA GCC CGA GCT GAT GCT GCC AAG GCC CTC GCT GAA GAA GCT A D Q L L A R A D A A K A L A E E A ~GCA AAG AAG A. K K
m CGG G
R
GAT ACC TTA CAA GAA GCT AAT GAC ATT CTC AAC AAC CTG D T L Q E A N D I L N N L
4611
1337
AAA GAT TTT GAT AGG CGC GTG AAC GAT AAC AAG ACG GCC GCA GAG GAG GCA CTA K
D
F
D
R
R
V
N
D
N
K
T
A
A
E
E
A
L
AGG AAG ATT CCT GCC ATC AAC CAG ACC ATC ACT GAA GCC AAT GAA AAG ACC AGA I? K I P A I N Q T I T E A N E K T F1
4119
1373
GAA GCC CAG CAG GCC CTG GGC AGT GCT GCG GCG GAT GCC ACA GAG GCC AAG AAC E A Q Q A L G S A A A D A T E A K N AAG GCC CAT GAG GCG GAG AGG ATC GCG AGC GCT GTC CAA AAG AAT GCC ACC AGC K A H E A E R I A - S A V Q K N A T S
4227
14G9
ACC AAG GCA GAA GCT GAA A& ACT TTT GCA GAA GTT ACA GAT CTG GAT AAT GAG T K A E A E R T F A E V T D L D N E GTG AAC AAT ATG TTG AAG CAA CTG CAG GAA GCA GAA P.AA GAG CTA AAG AGA PM V N N M L K Q L Q E A E K E L K R K
4335
1445
CAA GAT GAC GCT GAC CAG GAC ATG ATG ATG GCA GGG ATG GCT TCA CAG GCT K T Q D D A D Q D M M N A G M A S Q A A CAA GR4 GCC GAG ATC AAT GCC AGA AAA GCC AAA AAC TCT GTT ACT AGC CTC TYC Q E A 2 I N A R K A K N S V T S L i
4fWZ
1461
ACX ATT ATT Pp.T GAC CTC CTG GAG CAG CTG GGG CAG CTG GAT ACA GTG GAC CTG S I I :ID L L E Q L G Q L D T V D i AAT AAG CTA M.C (GAG ATT GAA GGC ACC CTA AAC ilAA GCC AAA GAT GAA ATG M G N K L N E I E G T L N K A K D E M i(
4551 -151-7
GTC AGC - GAT CTT GAT AGG AAA GTG TCT GAC CTG GAG AAT GAA GCC AAG AAG CAG V S D L D R K V S D L E N E A K K Q GAG GCT GCC ATC ATG GAC TAT AAC CGA GAT ATC GAG GAG ATC ATG AAG GAC AI'T E A A I M D Y N R D I E E I M K D I
2GC PAT CTG GAG r&C R
N
L
S
D
465'3 1553
ATC AGG AAG ACC TTA CCA TCT GGC TGC T C AAC ACC CCG I R K T L P S G C F N T P
TCC ATI' GAA AAG CCC TAG TGT S I E K P *
CTTTAGGGCT
C.4GGGGGGCA
GTTGTGAGGC CACAGAGTGC
CCACTCCTCT
GCTGCTATGG - - ATGC -
GGAACGCAGC
ATCCCTCTGA 1576 --
CTTGACACAA AGATTACATT
TTTCAGACX
Figure 2 Nuc kotide and deduced amino acid sequences of human laminin B2 cham pLB2 clone First line, nucleotide sequence of the P L B L clone, hecond line, deduced amino acid sequence The single termination codon I S indicated by an asterisk and the differences found In the nucleotide and amino acid sequences when compared to the published ones, are underlined
Mitochondrial DNA Downloaded from informahealthcare.com by University of Newcastle on 01/02/15 For personal use only.
H U M A N LAMININ B2 SEQUENCES
nick translated clone pLB2 as a probe. These two transcripts could result from differential splicing mechanisms leading to multiple mRNA species from a particular gene or even differential termination of transcription by the presence of more than one termination signal. DNA sequencing reactions, performed by the chain termination procedure of Sanger et a/. (1977) was determined on both strands and revealed that the clones isolated from the human umbilical vein endothelial cDNA library encode the carboxy terminal amino acid residues of the B2 chain of laminin (fig. 2). This sequence is homologous at the amino acid level to those of the corresponding human placenta (Pikkarainen et a/., 19881, human lung (Mattei et a/., 1988) and mouse (Sasaki and Yamada, 1987) B2 laminin chains. It i s interesting to note that there is identity between our clone, the placental and the mouse B2 cDNA clones concerning serine at position 1519. Thus lung B2 laminin differs from the above since a G+A change has replaced this serine by asparagine in the latter clone. O n the other hand, comparing our clone to the placental one, we have found one silent nucleotide change (G4200 instead of A) relative to placental laminin B2. Other differences relative to the latter, have been found in the 3’untranslated region. Thus, G4841, C4843, C4844, C4847 and A4848 in the placental clone have been substituted in our clone by A, G, G, G and C respectively. Those five-nucleotide substitutions clustered at the end of the sequence can be interpreted as cloning artefacts, frequently seen near the cloning site. O n the
277
other hand, we may consider the two nucleotide differences in the translated region as being due to tissue specific differential splicing, or to polymorphism among populations. We thank Dr. Ginsburg for providing us with the endothelial cDNA library and Maria de Lourdes Junqueira for the skilled technical assistance. The laminin B2 sequence has been deposited with the EMBL data library under the accession number x13939. (Received 5 April 1990)
REFERENCES Engel, I., Odermatt, E., Engel, A., Madri, J.A., Furthmayr, H., Rohde, H . and Timpl, R. (1981). Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. 1. Mol. Bio/., 150, 97-120. Ginsburg, D., Handin, R.J., Bonthron, D.T., Donlon, T.A., Bruns, G.A.P., Latt, S.A. and Orkin, S.H. (1985). Human von Villebrand factor (VWF): Isolation of complementary DNA (cDNA) clones and chromosomal localization. Science, 228, 1401-1 406. Martin, G.R. and Timpl, R. (1987). Laminin and other basement membrane components. Annu. Rev. Cell. Biol., 3, 57-87. Mattei, M.C., Weil, D., Pribula-Conway, D., Bernard, M.P., Passage, E., Van Cong, N’G., Tirnpl, R. and Chu, M. (1988). cDNA cloning, expression and mapping of human laminin B2 gene to chromosome lq31. Hum. Genet., 79, 235-241. Pikkarainem, T., Kallumki, T. and Tryggwason, K. (1988). Human laminin B2 chain. 1. Biol. Chem., 263, 6751-6758. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Roc. Nat. Acad. Sci. U S A, 74, 5463-5467. Sasaki, M. and Yamada, Y. (1987). The laminin 82 chain has a multidomain structure homologous to the B1 chain. /. BIo/. Chem., 262, 17111-17117. Timpl, R. and Martin, G. (1982). Components of basement membranes. In Furthmayer H. (ed.) lmmunochemistry of the Extracellular Matrix (Florida: CRC f’ress), vol. 2, pp.l 1%150.
0 1991 Harwood Academic
Publishers GmbH Printed in the United Kingdom
SHORT COMMUNICATION
Differences in human laminin B2 sequences Mitochondrial DNA Downloaded from informahealthcare.com by University of Newcastle on 01/02/15 For personal use only.
CECILIA L.S. SANTOS, JORGE SABBAGA and RICARDO BRENTANI Ludwig Institute for Cancer Research Brazil
- Sao Paul0 Branch,
Rua Prof. Antonio Prudente, 109, 4 andar, 01509 Sao Paulo,
E M B L Data Library Accession No. X13939.
some clones were established in E . coli RY 1089 cells and gel1 electrophoretical analyses of the corresponding protein extracts showed that P-galactosidase fusion proteins were synthesized by the induced lysogens. The recombinant protein from one lysogen (pLB2) was electroeluted from preparative gels and used to raise antibodies in mice. Western blot analysis revealed that the anti-recombinant protein pLB2 antiserum recognizes laminin B chains both frorn EHS mouse and from human placenta (fig. 1A). Northern blot analysis (fig. 16) on RNA isolated from HT 1080 human fibrosarcoma cells and rnouse placenta showed two bands of 5,5 and 7,5 kb using
A cDNA clone encoding the B2 chain of laminin has been isolated from a human endothelial A g t l l cDNA library. The nucleotide and deduced amino acid sequences of the clone were determined and showed one amino acid substitution (Ser1519 instead of Asn) when compared to lung laminin 62 chain and one silent nucleotide change (C4200 instead of A) in relation to the human placenta laminin 62 chain. Other differences in the 3’untranslated region were also found.
Key words: basement membrane, extracellular matrix, dothelial cell, laminin, laminin 62 chain
en-
Laminin, a major component of basement membranes, is a large glycoprotein consisting of three distinct polypeptide chains (A= 400kD; B1 = 210kD; B2= 200kD) linked together by disulphide bonds in a cross-shaped structure (Engel et a/. 1981; Timpl and Martin 1982). This glycoprotein has many biological activities such as promoting cell adhesion, migration, mitogenesis, growth and differentiation. Some of these properties are the result of interactions with other components of basement membranes which associate with different domains of the laminin molecule (Martin and Timpl 1987). The isolation of basement membrane genes will provide an opportunity to study the molecular mechanisms of these complex processes. For this purpose two 3, g t l l cDNA (EC-1: 3’oligo dT- and EC-2: hexanucleotide random primed) libraries (a gift from Dr. Ginsburg, University of Michigan), constructed using mRNA from human umbilical vein endothelial cells (Ginsburg et a/., 1985) were immunoscreened with an affinity purified polyclonal rabbit antiserum directed against mouse laminin extracted from the Engelbreth-Holm-Swarm (EHS) tumor. Positive clones showed cDNA inserts ranging from 600 to 3000 bp. Southern blot experiments showed that the clones isolated from the EC-1 library cross-hybridize with those isolated from the EC-2 library. Lysogens of
kD
A
B 1
2
3
1
kb 2
400-
- 7.5
200-
- 5.5
Figure 1 , (A) lmmunoreaction of antibodies to recombinant protein pLB2 with laminin chains. Laminin preparations isolated from human placenta and EHS mouse tumor were analysed by SDS-PACE, transferred to nitrocellulose filter and visualized with anti recombinant pLB2 antiserum. Lane 1 : mouse laminin; lane 2: human placenta laminin; lane 3: pLB2 recombinant protein. Molecular markers are shown on the left; (B) Northern blot analysis. Total RNA (20ug) from mouse placenta (lane 1) and human HT 1080 fibrosarcoma cells (lane 2) were hybridized with nick translated pLB2 clone. Transcript sizes, given in kilobase pairs (kb) were estimated by comparison with the positions of 28s and 18s ribosomal RNA on the gel.
275
276
C . L. S. SANTOS ETAL.
A CTG GAG AAT GAA GCA AAT AAC ATA AAG ATG GAA GCT GAG AAT ZTG GAA CAA L E N E A N N I K M E A E N L E Q
37%
CTG ATT GAC CAG AAA TTA AAA GAT TAT GAG GAC CTC AGA GAA GAT ATG AGA GGG i I D Q K L K D Y E D L R E D M R G AAG K
1;AA
E
CTT GAA GTC AAG AAC CTT CTG GAG AAA GGC AAG ACT GAA CAG CAG ACC L E V K N L L E K G K T E Q Q T
30G3
1361
Mitochondrial DNA Downloaded from informahealthcare.com by University of Newcastle on 01/02/15 For personal use only.
GCA GAC CAA CTC CTA GCC CGA GCT GAT GCT GCC AAG GCC CTC GCT GAA GAA GCT A D Q L L A R A D A A K A L A E E A ~GCA AAG AAG A. K K
m CGG G
R
GAT ACC TTA CAA GAA GCT AAT GAC ATT CTC AAC AAC CTG D T L Q E A N D I L N N L
4611
1337
AAA GAT TTT GAT AGG CGC GTG AAC GAT AAC AAG ACG GCC GCA GAG GAG GCA CTA K
D
F
D
R
R
V
N
D
N
K
T
A
A
E
E
A
L
AGG AAG ATT CCT GCC ATC AAC CAG ACC ATC ACT GAA GCC AAT GAA AAG ACC AGA I? K I P A I N Q T I T E A N E K T F1
4119
1373
GAA GCC CAG CAG GCC CTG GGC AGT GCT GCG GCG GAT GCC ACA GAG GCC AAG AAC E A Q Q A L G S A A A D A T E A K N AAG GCC CAT GAG GCG GAG AGG ATC GCG AGC GCT GTC CAA AAG AAT GCC ACC AGC K A H E A E R I A - S A V Q K N A T S
4227
14G9
ACC AAG GCA GAA GCT GAA A& ACT TTT GCA GAA GTT ACA GAT CTG GAT AAT GAG T K A E A E R T F A E V T D L D N E GTG AAC AAT ATG TTG AAG CAA CTG CAG GAA GCA GAA P.AA GAG CTA AAG AGA PM V N N M L K Q L Q E A E K E L K R K
4335
1445
CAA GAT GAC GCT GAC CAG GAC ATG ATG ATG GCA GGG ATG GCT TCA CAG GCT K T Q D D A D Q D M M N A G M A S Q A A CAA GR4 GCC GAG ATC AAT GCC AGA AAA GCC AAA AAC TCT GTT ACT AGC CTC TYC Q E A 2 I N A R K A K N S V T S L i
4fWZ
1461
ACX ATT ATT Pp.T GAC CTC CTG GAG CAG CTG GGG CAG CTG GAT ACA GTG GAC CTG S I I :ID L L E Q L G Q L D T V D i AAT AAG CTA M.C (GAG ATT GAA GGC ACC CTA AAC ilAA GCC AAA GAT GAA ATG M G N K L N E I E G T L N K A K D E M i(
4551 -151-7
GTC AGC - GAT CTT GAT AGG AAA GTG TCT GAC CTG GAG AAT GAA GCC AAG AAG CAG V S D L D R K V S D L E N E A K K Q GAG GCT GCC ATC ATG GAC TAT AAC CGA GAT ATC GAG GAG ATC ATG AAG GAC AI'T E A A I M D Y N R D I E E I M K D I
2GC PAT CTG GAG r&C R
N
L
S
D
465'3 1553
ATC AGG AAG ACC TTA CCA TCT GGC TGC T C AAC ACC CCG I R K T L P S G C F N T P
TCC ATI' GAA AAG CCC TAG TGT S I E K P *
CTTTAGGGCT
C.4GGGGGGCA
GTTGTGAGGC CACAGAGTGC
CCACTCCTCT
GCTGCTATGG - - ATGC -
GGAACGCAGC
ATCCCTCTGA 1576 --
CTTGACACAA AGATTACATT
TTTCAGACX
Figure 2 Nuc kotide and deduced amino acid sequences of human laminin B2 cham pLB2 clone First line, nucleotide sequence of the P L B L clone, hecond line, deduced amino acid sequence The single termination codon I S indicated by an asterisk and the differences found In the nucleotide and amino acid sequences when compared to the published ones, are underlined
Mitochondrial DNA Downloaded from informahealthcare.com by University of Newcastle on 01/02/15 For personal use only.
H U M A N LAMININ B2 SEQUENCES
nick translated clone pLB2 as a probe. These two transcripts could result from differential splicing mechanisms leading to multiple mRNA species from a particular gene or even differential termination of transcription by the presence of more than one termination signal. DNA sequencing reactions, performed by the chain termination procedure of Sanger et a/. (1977) was determined on both strands and revealed that the clones isolated from the human umbilical vein endothelial cDNA library encode the carboxy terminal amino acid residues of the B2 chain of laminin (fig. 2). This sequence is homologous at the amino acid level to those of the corresponding human placenta (Pikkarainen et a/., 19881, human lung (Mattei et a/., 1988) and mouse (Sasaki and Yamada, 1987) B2 laminin chains. It i s interesting to note that there is identity between our clone, the placental and the mouse B2 cDNA clones concerning serine at position 1519. Thus lung B2 laminin differs from the above since a G+A change has replaced this serine by asparagine in the latter clone. O n the other hand, comparing our clone to the placental one, we have found one silent nucleotide change (G4200 instead of A) relative to placental laminin B2. Other differences relative to the latter, have been found in the 3’untranslated region. Thus, G4841, C4843, C4844, C4847 and A4848 in the placental clone have been substituted in our clone by A, G, G, G and C respectively. Those five-nucleotide substitutions clustered at the end of the sequence can be interpreted as cloning artefacts, frequently seen near the cloning site. O n the
277
other hand, we may consider the two nucleotide differences in the translated region as being due to tissue specific differential splicing, or to polymorphism among populations. We thank Dr. Ginsburg for providing us with the endothelial cDNA library and Maria de Lourdes Junqueira for the skilled technical assistance. The laminin B2 sequence has been deposited with the EMBL data library under the accession number x13939. (Received 5 April 1990)
REFERENCES Engel, I., Odermatt, E., Engel, A., Madri, J.A., Furthmayr, H., Rohde, H . and Timpl, R. (1981). Shapes, domain organizations and flexibility of laminin and fibronectin, two multifunctional proteins of the extracellular matrix. 1. Mol. Bio/., 150, 97-120. Ginsburg, D., Handin, R.J., Bonthron, D.T., Donlon, T.A., Bruns, G.A.P., Latt, S.A. and Orkin, S.H. (1985). Human von Villebrand factor (VWF): Isolation of complementary DNA (cDNA) clones and chromosomal localization. Science, 228, 1401-1 406. Martin, G.R. and Timpl, R. (1987). Laminin and other basement membrane components. Annu. Rev. Cell. Biol., 3, 57-87. Mattei, M.C., Weil, D., Pribula-Conway, D., Bernard, M.P., Passage, E., Van Cong, N’G., Tirnpl, R. and Chu, M. (1988). cDNA cloning, expression and mapping of human laminin B2 gene to chromosome lq31. Hum. Genet., 79, 235-241. Pikkarainem, T., Kallumki, T. and Tryggwason, K. (1988). Human laminin B2 chain. 1. Biol. Chem., 263, 6751-6758. Sanger, F., Nicklen, S. and Coulson, A.R. (1977). DNA sequencing with chain-terminating inhibitors. Roc. Nat. Acad. Sci. U S A, 74, 5463-5467. Sasaki, M. and Yamada, Y. (1987). The laminin 82 chain has a multidomain structure homologous to the B1 chain. /. BIo/. Chem., 262, 17111-17117. Timpl, R. and Martin, G. (1982). Components of basement membranes. In Furthmayer H. (ed.) lmmunochemistry of the Extracellular Matrix (Florida: CRC f’ress), vol. 2, pp.l 1%150.
