Appendix
APPENDIX
Appendix 1: The pcDNA3.1(serglycin)/Myc-His-vector
Colour codes: CMV-promoter region, GAG attachment site, Myc-epitope, His-flag, Polyadenylation signal.
GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCT
CTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTA
AGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGAT
GTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA
GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATT
GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGG
TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGC
CCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTAT
TACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCAC
CCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCAT
CAAT
TATA box
transcription start
TGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGC
TTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACGGGCCCTCTAGAGTT
924/1
954/11
ATG ATG CAG AAG CTA CTC AAA TGC AGT CGG CTT GTC CTG GCT CTT GCC CTC ATC CTG GTT
Met Met Gln Lys Leu Leu Lys Cys Ser Arg Leu Val Leu Ala Leu Ala Leu Ile Leu Val
984/21
1014/31
CTG GAA TCC TCA GTT CAA GGT TAT CCT ACG CAG AGA GCC AGG TAC CAA TGG GTG CGC TGC
Leu Glu Ser Ser Val Gln Gly Tyr Pro Thr Gln Arg Ala Arg Tyr Gln Trp Val Arg Cys
1044/41
1074/51
AAT CCA GAC AGT AAT TCT GCA AAC TGC CTT GAA GAA AAA GGA CCA ATG TTC GAA CTA CTT
Asn Pro Asp Ser Asn Ser Ala Asn Cys Leu Glu Glu Lys Gly Pro Met Phe Glu Leu Leu
1104/61
1134/71
CCA GGT GAA TCC AAC AAG ATC CCC CGT CTG AGG ACT GAC CTT TTT CCA AAG ACG AGA ATC
Pro Gly Glu Ser Asn Lys Ile Pro Arg Leu Arg Thr Asp Leu Phe Pro Lys Thr Arg Ile
1164/81
1194/91
CAG GAC TTG AAT CGT ATC TTC CCA CTT TCT GAG GAC TAC TCT GGA TCA GGC TTC GGC TCC
Gln Asp Leu Asn Arg Ile Phe Pro Leu Ser Glu Asp Tyr Ser Gly Ser Gly Phe Gly Ser
1224/101
1254/111
GGC TCC GGC TCT GGA TCA GGA TCT GGG AGT GGC TTC CTA ACG GAA ATG GAA CAG GAT TAC
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Phe Leu Thr Glu Met Glu Gln Asp Tyr
1284/121
1314/131
CAA CTA GTA GAC GAA AGT GAT GCT TTC CAT GAC AAC CTT AGG TCT CTT GAC AGG AAT CTG
Gln Leu Val Asp Glu Ser Asp Ala Phe His Asp Asn Leu Arg Ser Leu Asp Arg Asn Leu
1344/141
1374/151
CCC TCA GAC AGC CAG GAC TTG GGT CAA CAT GGA TTA GAA GGA ATT CCA CCA CAC TGG ACT
Pro Ser Asp Ser Gln Asp Leu Gly Gln His Gly Leu Glu Gly Ile Pro Pro His Trp Thr
1404/161
1434/171
AGT GGA TCC GAG CTC GGT ACC AAG CTT GGG CCC GAA CAA AAA CTC ATC TCA GAA GAG GAT
Ser Gly Ser Glu Leu Gly Thr Lys Leu Gly Pro Glu Gln Lys Leu Ile Ser Glu Glu Asp
1464/181
1494/191
CTG AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA GTTTAAACCGCTGATCAGCCTCGACTG
Leu Asn Ser Ala Val Asp His His His His His His * * *
I
Appendix
TGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGT
CCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG
GACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAA
GAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCG
CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCC
GGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAA
II
Appendix
Appendix 2: Incorporation of 35(S)sulphate
A parallel experiment to the 35(S)sulphate labelling of the transfected MDCK II subclones shown in
figure 4.12. Note that in this experiment, the cells were labelled after three days incubation on
Transwell polycarbonate filter membranes. This may give a different 35(S)macromolecule secretion
compared to four days growth on filter membranes.
The secretion of 35(S)macromolecules secreted from polarised cultured cells was analysed by
seeding MDCK II subclones into transwell polycarbonate filter membranes (24 mm) in complete
medium (method 3.4.2). After 3 days incubation, the cells were labelled with approximately 0.2
mCi/ml 35S(sulphate) for 20 hours. Apical and basolateral media were harvested separately, giving
1 ml apical and 2 ml basolateral medium. Unincorporated 35S(sulphate) was removed by
chromatography of 1 ml medium on Sephadex G-50 Fine (method 3.7.3). The capacity of each
subclone to incorporate 35S(sulphate) into macromolecules secreted apically and basolaterally was
analysed by counting 50 l of the material eluted form the Sephadex G-50 columns. The counted
values were used to determine the total amount of 35(S)macromolecules eluted from the Sephadex
G-50 Fine column (1.5 ml).
III
Appendix
Appendix 3: Test of PG binding to plasitc tubes
In some Hi-Trap chelating chromatography experiments, the recovery after 250 kDa PG
purification was much lower than expected. Only 20-50 percent of the labelling activity remained
after Hi-Trap chelating chromatography. The Hi-Trap chelating column was thus washed with
EDTA to strip the column for Ni2+ ions, to check if any labelled material still was bound to the
column matrix. The eluate contained only small amounts of 35(S)sulphate, and thus excluded this
possibility.
Another possibility was that the 35(S)proteoglycans were adhered to the collecting tube wall. The
high negative charge on the glycosaminoglycan chains can bind strongly to positive charges on the
plastic tube surface. To test this, 1 ml samples (5000 cpm), were mixed in eppendorp tubes,
transferred to the collecting tube and incubated for 20 minutes at room temperature. The samples
were then transferred to scintillation tubes and counted. As references, samples were mixed in
eppendorp tubes only, before transfer, and directly into the scintillation tubes.
All reference samples contained approximately 5000 cpm, but the samples from the tubes contained
only half of the activity. This explains the reduced recovery. The collecting tubes (Sarstedt,
Germany, No: 55.484) were thus replaced with eppendorp tubes in the following experiments.
IV
Appendix
Appendix 4: Calculation of GAG substitution on 250 kDa PG secreted
by TGF- stimulated subclone 1-7
Relative content of GAG
Peak 1
Chondroitin sulphate*
Heparan sulphate*
Elution after Hi-Trap**
CS-250 kDa PG in peak 1
HS-250 kDa PG in peak 1
Peak 2
Chondroitin sulphate***
Heparan sulphate***
Elution after Hi-Trap****
CS-250 kDa PG in peak 2
HS-250 kDa PG in peak 2
SUM peak 1 and peak 2
CS-250 kDa PG
HS-250 kDa PG
SUM secreted 250 kDa PG
Clone 1-7
Apical
Basolateral
10
10
90
90
15
20
1.5
2
13.5
18
Apical
Basolateral
85
85
15
15
27
22
23
19
4
3
Apical
Basolateral
24.5
21
17.5
21
42
42
Figure I: Secretion of CS- and HS-250 kDa PG to the apical and the basolateral
compartments.
Values are from: (*) Figure 4-28.C, (**) Table 4-11, (***) Figure 4-29.C and (****) Figure
4-27.D.
Relative contents of GAG
CS-250 kDa PG
HS-250 kDa PG
SUM secreted 250 kDa PG
Percent CS and HS
CS-250 kDa PG in compartment
HS-250 kDa PG in compartment
Elution after Anion exchanger
Distribution of produced 250 kDa PG
CS-250 kDa PG
HS-250 kDa PG
Clone 1-7
Apical
Basolateral
24.5
21
17.5
21
42
42
Apical
Basolateral
58 %
50 %
42 %
50 %
35 %
65 %
Apical
Basolateral
20 %
33 %
14 %
33 %
Figure II: Apical and basolateral secretion of CS- and HS-250 kDa PG across the cell layer.
V