Introduction
Materials and methods
Cloning and Construction of Matrilin-3 cDNAs
Primers | Primer Sequences (5'-->3') | PCR purpose | Results shown in Figures |
---|---|---|---|
1 | TAA TAC GAC TCA CTA TAG GG | T7, amplifying inserts from pCDNA3.1 | |
2 | AAG GAC GAT GAT GAC AAA GCT GCA AAT ACA TGT GCA CT | Adding a flag tag | |
3 | TGT CAT CAT CGT CCT TAT AGT CCC CCC AGA CTC CAC AGC T | ||
4 | GAG GAG AGG GTT AGG GAT AGG CTT A | Amplifying inserts from pCDNA3.1 | |
5 | ACT GCA AGC TGA GCA AGT CTT CTT G | Adding a vWFA domain into minimatn3 (combining with the PCR product of primers 1 and 4) | Figure 4 |
6 | ATCTGC GTT AGA GCC ACA ACA AGC AGT | ||
7 | ACT GCA AGC TGA GCA AGT CTT CTT G | Replacing minimatn3 coiled-coil domain with that of matn1 (combining with the PCR product of primers 1 and 4) | Figure 7 |
8 | ATC TGC GTT AGA GCC ACA ACA AGC AGT | ||
9 | AAA GAA CAA CCT GGG TGG CAG TCA TGA | Introducing R116W mutation in the vWFA domain | Figure 2 |
10 | TCA TGA CTG CCA CCC AGG TGG TTC TTT | ||
11 | GAT GAC AAA GCA CCT CCT CAG CCC AGA | Adding a flag tag | |
12 | ATC TTC CTC ACT GCA GGT CTT CCC ATC ATT | ||
13 | ACC TGC AGT GAG GAA GAT CCA TGC GAA TGT | Creating Δmatn1 by deleting vWFA2 | Figure 5 |
14 | ACC TGC AGT TGC GAA TGT AAA TCT ATA GT | ||
15 | ACA TTC GCA ACT GCA GGT CTT CCC ATC AT | Creating Δmatn1_del by deleting 4 amino acids from Δmatn1 | Figure 6 |
16 | ACT TGC TCA GCT TGC AGT GGT GGG TCA | ||
17 | TGT GGC TCT AAC GCA GAT TTT CAT TTG | Amplifying mantn1 coiled-coil domain | |
18 | ACT TGC TCA GCT GTC AGT GGT GGG TCA | ||
19 | TCT GGC TCT AAC GCA GAT TTT CAT TTG | Amplifying mantn1 vWFA2 domain |
Transfection of Matrilin cDNAs
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Protein Pulse-Chase
Immunohistochemistry
Results
MED mutation in the vWFA domain of MATN3
Insertion of vWFA2 domain into MATN3
Deletion of vWFA2 domain from MATN1
Proteolysis of matn1
Exchange of the coiled-coil domain between MATN1 and MATN3
Discussion
Secretion
Assembly
Proteolysis
Matrilin | Species | Amino Acid Sequence | Number (a.a.) |
---|---|---|---|
MATN1 | Human | EE DP | 4 |
Mouse | EE DP | 4 | |
Chicken | EE DP | 4 | |
MATN2 | Human | KLKKGICEALEDSDGRQDSPAGELPKTVQQPT ESEPVTINIQDLLSCSNFAVQHRYLFEE DNLL RSTQKLSHSTKPSGSPLEE | 83 |
Mouse | KLKEGICEALEDSGGRQDSAAWDLPQQAHQP TEPEPVTIKIKDLLSCSNFAVQHRFLFEE DN LSRSTQKLFHSTKSSGNPLEE | 83 | |
Chicken | ELKVQICEALRNSAHQQHLSSGRLHRTNPQPSGPESTTVEITDVLACPSLAIQHKYLFEDSQSHSTRTTAKT | 72 | |
MATN3 | Human | ATEE ARRLVSTEDA | 14 |
Mouse | DIEE ARSLISIEDA | 14 | |
Chicken | RATTSSLVTDEE A | 13 | |
MATN4 | Human | PEE GISAGTELRSP | 14 |
Mouse | PEE GIGAGTELRSP | 14 | |
Chicken | PEE GRGETEIRSP | 13 |