E HPT AD R AGCS AS GAC Y S L HHAT MKRQ AAE E AC I L R G GAL S T VRAG A. THBD. F P AP AE P QP G GS QCVE HDC F AL Y P GP AT F L NAS Q I ...
MMRN2FL
Mock
MMRN2FL
Mock
stacking gel
stacking gel
kDa
kDa 250
Probe: CLEC14A-ECD-Fc
250
Anti His
Figure S1. CLEC14A-ECD binds MMRN2 under non-reduced conditions. HEK293T transfected with MMRN2FL were lysed and separated on a 6% polyacrylamide gel under nonreducing conditions. MMRN2FL forms large molecular weight complexes that barely enter the stack gel. CLEC14A far westerns show binding to MMRN2FL multimers. His tag blot is included to show expression of MMRN2FL.
Human Bull Chimp Rat Mouse Zebrafish (a) Zebrafish (b) Chicken Consistency
495 492 495 490 495 615 523 526
QK L Y L DL DV I QK L Y F D L D V I QK L Y L D L D V I R R V Y S D MD V I QR V NS DV DV A I AL GAT V T H L K V I NS S L V R L QK I P S D A N T L 458357655 7
R E G QR D A T R R E D QR D T T R R E G QR D A T R P E G R R D AM H P E G H R D VM H E QT V A D VM K E K E V DNV T Q E E DL K N I T Y 584467665
Human Bull Chimp Rat Mouse Zebrafish (a) Zebrafish (b) Chicken Consistency
561 558 561 554 561 685 591 581
T S R L R S QV Q A A A R L R S QL R A T S R L R S QV Q A R A RM R S QL R A R A R I R S QL R A V T QA L A S L L G I L QL QD S L L D N R QL QL QV A S 357766774 6
Human Bull Chimp Rat Mouse Zebrafish (a) Zebrafish (b) Chicken Consistency
631 628 631 624 631 755 661 651
PLSYEQI RV A PLRYEQI RV A PLSYEQI RV A P L NY E Q I R L A P L DY E Q I R L A R F S I P QL D E R DL S L P DL L Q T I S S V V QL QE F 466658854 5
L E E T QV S L D E L E E T QV T L D E L E E T QV S L D E L E E T QV S P D E L E E T QV S L D E V N E N I L A QE M A R QN S L A L E E L E G QQS K D V K 777666646 7
R R - - - - QL D G R R - - - - QQD G R R - - - - QL D G QH - - - - QL - D QH - - - - QL D G MA N R H N R A W V A DL E - - R NE A - - - - - - QL E S 430000854 4
S S L QA L QN A S S L QA L R D T S S L QA L QN A G S L QA L QS T S S L QA L QS T P S V E DL K L G T E ME D L QH G V F K DL L R N E 4 6 6 7 5 *7 4 4
V V V V V L L I 8
DA V S L A V DA A S L S L A V DT DA V S L A V DA D A MS S A L D A D A MS S A MD A L N V QN S L A F L N MK E S L A F E E L S S AF P F 457748655
H Q H Y Y Q E I 4
K AE GE R AR A QA E G E R A R A K AE GE R AR A R GE GE R AR A R GE GE R AR A QE K S R T L QH QG R S R S L QD H QS L N L R QE 656565675
A E A E E N S E 5
L DDE V GA L K A L GGE V S AL Q A L DDE V GA L K A L DHA V E A L K T L DHA V E A L K T S QQD I E M L Q K S K L E I QG L R E F S E D I GL L K K 5 5 3 5 9 4 5 *7 5
A A A E A R HE V AE T E I R R D I A A A E A R HE V A A NGT R K E I A A NGT R K E I QD R A K V E K I QD G E QS A E I K DE E I HR H I 654545469
R R R R R K Q K 7
QL H S A F A A QL H S S F S A QL H S A F A A L L HGS F A A L L HGS F T A QL E G T F N T GL S AAF S S Y L NS S F GS 4 *6 6 7 *5 7
L L L L L L L L *
E DA L R HE A V E DA L R HE A V E DA L R HE A V E D A L R H QA V E D A L R H QA V QD A T R H S D V NDA V R HS E V E D AM R H E A A 7 * *7 * *6 6 8
L L L L L L L L *
AAL F GE E V L E AAL F GE E V ME AAL F GE E V L E A A L F G E E MV D AAL F GE E MI D E I L L GE E V L E E V L L GE E V L E E A L L GE E FM E 6 7 *7 * * *6 7 8
E MS E QT P G P E MS E E G P G T E MS E QT P G P E MS E E P P R P E MS E E A P R P F T K WS QQD K F T H WS QS R Q V L F E E DP S T 465663644
L L L L L R K L 6
L QD A A S G L Q E L MD A A S G L Q E L QD A A S G L Q E L QD A A S G L Q E L QD A A S G L Q E MK D MQK Q I N G I QMMQQK I E T L K E I S NK L Q N 867666587 5
QA L G WD E L A QA L G WD A L A QA L G WD E L A QA V G WE D L A QA I G WE D L A HS L S L A SML HDR S L T S L R QN V T L E S L V 766655596
A A A T T N R K 4
R - V T A L E QA S R - V T A L E QA S R - V T A L E QA S R - V E AL E K A A R - V E AL E K A A A - A T P QV T A A K H P D G D E MN S R - L HHL E R G Q 706556856 6
- -EPPR - - - - GAR R - - - -EPPR - - - - GGF V - - - - GGF V - - DE P S L L - - DDP V P Y - - QN N H H V H A S 003324000
S 0
-P - - - - - - - - QT - - - - - - -P - - - - - - - - E QH P R - - - - E QH P QL - - - A D WA S T G T K - S E K P G MK Q Q K R P E QE T QT L 042111100 0
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - RKRE - - - - MK G T - - - - S ML D E A N QL 000000000
S 0
A A A T T T L S 5
L L L L L L L L *
Unconserved 0 1 2 3 4 5 6 7 8 9 10 Conserved
Figure S2. Amino acid sequence alignments of human MMRN2495-674 and corresponding regions from different species. PRALINE (1) generated amino acid alignments of MMRN2 regions from human (Accession no. Q9H8L6), bull (E1BJW3), chimp (H2Q273), rat (D4ABX6), mouse (A6H6E2), chicken (E1BTB7) and two zebrafish proteins MMRN2a (zebrafish (a)) (F1R6V5) and MMRN2b (zebrafish (b)) (E7FG77). This reveals a highly conserved region between residues 588-620 (red line) of human MMRN2. This region contains 72% amino acid identity between human and mouse MMRN2. Residues are labelled for conservation by colour. Blue = unconserved, red = conserved.
1. Simossis VA, Heringa J. PRALINE: a multiple sequence alignment toolbox that integrates homology-extended and secondary structure information. Nucleic Acids Res 2005; 33: W289-W294.
kDa 250
130 100 70
130 100 70
55
55
35
35
25
25
non-reduced
reduced
Probe: CLEC14A-ECD-Fc
non-reduced
MMRN2495-674
MMRN2487-674
MMRN2495-674
MMRN2487-674
MMRN2495-674
MMRN2487-674
MMRN2495-674
MMRN2487-674 kDa 250
reduced Anti His
Figure S3. CLEC14A-ECD-Fc binds MMRN2487-674 and MMRN2495-674. Each MMRN2 fragment was transfected into HEK293T, lysed and subjected to far western blotting using CLEC14A-ECD-Fc, this revealed binding to both fragments under reduced (R) and nonreduced (NR) conditions. Under NR conditions MMRN2487-674 forms large molecular weight multimers that also bind CLEC14A. His tag blot included to show expression of each protein.
70
mMMRN2495-678
hMMRN2495-674
mMMRN2495-678
kDa 70
hMMRN2495-674
mMMRN2495-678
hMMRN2495-674
mMMRN2495-678
hMMRN2495-674
kDa
55
55
35
35
25
25
non-reduced
reduced
Probe: mCLEC14A-ECD-Fc
non-reduced
reduced
Anti His
Figure S4. Mouse CLEC14A directly binds to human MMRN2495-674 and mouse MMRN2495. Far western blots using mouse CLEC14A-ECD-Fc shows specific binding to human
678
MMRN2495-674 and equivalent mouse MMRN2495-678, under reduced and non-reduced conditions. His tag blot is included to show expression of each protein fragment. Mouse MMRN2495-678 likely appears at a higher molecular weight due to a potential glycosylation site within this region.
β0
CLEC14A THBD CD248 CD93 Consistency
22 17 18 24
- - - E HP T A D R F P A P A E P QP G - QD P WA A E P - - - - - - A DT E 000201444 1
AGC S AS GAC G S QC V E H D C R AAC GP S S C AV V C V GT AC 43364335 *
α2
CLEC14A THBD CD248 CD93 Consistency
79 77 76 78
136 128 123 134
Y F Y Y 8
AGP G - - P GG GDG - - - - - AGP - - - - - QL L R R E A A L 422000000
G G T 2
R S - - CT AR R NGA P L C GP L G G - P C P A QR NS - - C I S K R 460162546
S K D L L F W VA V GR R R L W IG - AS R L L W IG A R MS K F W I G 2 3 3 4 4 6 *9 7
S L H H A T MK R Q AL Y P GP AT F L AL F P R R R T F L T A HS GK L S A A 665343353 4
α2
AAE E AC I L R NA S Q I C DGL E A WR A C R E L E A QN H C N QN 4 *3 4 3 *2 2 3
G R G G 6
L1
L L L L *
β3
C C C C *
α1
GAL S T V R AG G H L MT V R S S GDL A T P R T P GNL A T V K S K *3 *5 *6 8 6 3
A V E E 4
E L R AV L AL L A A DV I S L L L E A QR V D S L V E A QH V QR V L 665293388
R N G A 3
(Long loop)
β2
L4
CLEC14A THBD CD248 CD93 Consistency
β1
E R R R S HC T L E QL P P G C G D P K QR QA R QC QL Q QR E K G K C L D P 8643425224
L2
N - E P L R GF S W R L GP L R GF Q W R - - P L R GF T W S - L P L K GF S W 5 0 0 * *8 * *5 *
β4
A V L QA T G - - - - - G V E P A G W K V A V S A A E A T - - - V P S E P I WE V A L E - - - - - - - - A S GE HR WL V S L L L DL S Q P L L P S R L P K WS 758321010 0 00233332 * 3
L S S DP GGL E S V - - - T GDNN T T - - - T G D QD T V - - - GGGE D T 5 0 0 0 3 *5 3 6 7
L3
D T L QWV E E P Q S Y S R WA R L D L A F T N WA QP A S P Y S N WH K E L R 3 4 5 5 *4 5 3 2 3
β5
E MR C H L R A N G E QQC E V K A D G E GS C T L AV D G E GP C G- - S P G *3 3 *2 3 2 5 4 *
YL CKY F L C E F Unconserved 0 1 2 3 4 5 6 7 8 9 10 Y L C QF S P GS N Conserved 45554
Figure S5. Amino acid sequence alignments of CTLD group 14 family members. Human CLEC14A (Accession no. Q86T13), CD93 (Q9NPY3), THBD (P07204) and CD248 (Q9HCU0) CTLD sequences were aligned using PRALINE without inclusion of signal peptides. The αhelices (yellow rectangle) and β-sheets (green arrow) are mapped onto the alignment corresponding to their position within the predicted CLEC14A-CTLD model. There are also four predicted loops L1-L4 mapped within the long-loop region (red) as described for tetranectin CTLD. The L1 loop contains the 97-108 region and the non-canonical cysteine 103. The L4 contains the other non-canonical cysteine C138. Each cysteine residue within the longloop region is labelled with a black dot.
MMRN2495-603
MMRN2495-674
MMRN2495-603
MMRN2495-674
Marker
kDa
100 70 55 35 25
15
10 reduced
non-reduced
Figure S6. Coomassie stain of E.coli expressed and purified MMRN2495-674 and MMRN2495-603. SDS-PAGE and coomassie staining reveals both recombinant protein preparations are of high purity. MMRN2495-674 resolves at ~20 kDa and MMRN2495-603 at ~12.5 kDa.
LLC Negative 104
4 10
LLC mFC 104
4 10
0.05%
3 1010
3 1010
3
GFP GFP
GFP
GFP
3
2 1010
2 1010 2
1 1010
0 1010
0 1010
1
1
0
0
0
0
200 200
400 400
600 600
FSC-H FSC-H
800 800
1K
1000
LLC mCLEC14A mFc
4 10
01 LLC -ve.001 Ungated 97.31% 20328
104
4 10
3 10 10
3 1010
2 10 10
2 1010
GFP
GFP
GFP
2
200 200
400 400
600 600
FSC-H FSC-H
800 800
1000 1K
LLC m495-678 mFc
02 LLC mFc.002 Ungated 93.49% 20769
2
1 10 10
1 1010
0 10
0 1010
1
1
10
00
3
3
GFP
2
1 1010
104
97.38%
0
0
00
200 200
400 400
600 600
FSC-H FSC-H
800 800
03 LLC mC14A mFc.003 Ungated 20995
1000 1K
0
0
200 200
400 400
600 600
FSC-H FSC-H
800 800
1000 1K
04 LLC m495-678 mFc.004 Ungated 20698
Figure S7. Flow cytometry analysis of Lewis lung carcinoma transductants. Levels of green fluorescent protein (GFP) were compared to non-transduced cells (LLC –ve). All transduction efficiencies were over 90%.
Cell count
600000
ns
ns
400000
200000
0
mFc
mCLEC14A mFc
m495-678 mFc
LLC Transductants
Figure S8. Cell proliferation is unaffected between Lewis lung carcinoma transductants in vitro. Cells were plated and allowed to proliferate for 2 days and then counted using a coulter counter. This experiment was performed three times. There was no large difference in the cell count between each LLC transfectant. Mann Whitney test, mFc vs. mC14A mFc p = 1.0, mFc vs. m495-678 p = 0.7 n=3. Error bars represent SEM.
Figure S9. CD31+ vessel analysis. (A) For each image CD31 staining thresholds were set using an automated Triangle thresholding method using ImageJ Fiji. (1). (B) The subsequent binary image was then analysed using the ‘Particle Analysis’ plugin to measure area, average ‘vessel’ size, and percentage area. (C) The ImageJ macro that was used is displayed.
1.
Zack GW, Rogers WE, Latt SA. Automatic measurement of sister chromatid exchange frequency. Journal of Histochemistry and Cytochemistry 1977; 25: 741–753.
MMRN2 fragment
CLEC14A binding
CD93 binding
CD248 binding
MMRN2FL
+
+
+
MMRN2EMI-CC
+
Not tested
+
MMRN2CC-C1q
+
Not tested
+
MMRN2CC
+
Not tested
+
MMRN2133-486
-
Not tested
+
MMRN2487-820
+
Not tested
-
MMRN2487-674
+
Not tested
-
MMRN2495-674
+
+
-
MMRN2495-603
-
-
-
MMRN2530-624
+
Not tested
-
MMRN2604-674
-
Not tested
-
Supplementary Table 1. A summary of which CTLD group 14 family members bind to which regions of MMRN2.