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清道夫受体介导的脂质蓄积的分子机制

Intracellular Accumulation of Lipids via Scavenger Receptors
Qi Chen
Nanjing Medical University
2005.8
Formation of foam cell
    Athersclerosis is a chronic inflammatory reaction caused by macrophages and lymphocytes that uptake the pathogenicity  lipids invading the vessel.
SR-A and CD36 are the principal receptors responsible for foam-cell formation


Macrophage cell takes up lipid by SR-A
Structure of  SR-A
   Six domains

   Three types

   Trimeric molecule
  Foam cell formation
  Mφ adhesion
  Host defense and innate 
    immunity
  Adaptive responses
  Disorders of the nervous
   system
  Phagocytosis  of  apoptotic  
    cells
Functions of  SR-A
The cytoplasmic domain is the key part of the receptor???
 mediate the internalization of ligands such as ox-LDL
  participate the regulation of cell adhesion and migration
  accommodate the expression and transport of receptor
  link-coupled with downstream of signaling pathway
Functions of SR-A cytoplasmic domain:
    Cytoplasmic domain:
 

 Murine: MTKEMTENQRLCPHEREDADCSSESVKFDARSMTASLP
                 HSTKNGPSVQEKLKSFK (55)
 Rabbit:  MAQWDSFTDQQEDTDSCSESVKFDARSNTALLPPNPK
                 GPPLQEKLKSFK (50)
 Bovine: MAQWDDFPDQQEDTDSCTESVKFDARSVTALLPPHPKN
                 GPTLQERMKSYK (50)
 Human: MEQWDHFHNQQEDTDSCSESVKFDARSMTALLPPNPK 
                 NSPSLQEKLKSFK (50)

  phosphorylation of cytoplasmic domain

  signal sequence in cytoplasmic domain

  interactors with cytoplasmic domain

Focus on cytoplasmic domain:
Bovine Model
Human Model
Murine Model
Rabbit  Model
Ser21(rat)
increases expression of receptor
promotes metabolism of lipid

Fong LG et al. J Biol Chem, 274, 36088-36816 (1999)

phosphorylation of cytoplasmic domain
phosphorylation of cytoplasmic domain
Ser49(rat)
reduces uptake of lipid
Fong LG et al. J Biol Chem, 274, 36088-36816 (1999)

FDARS 
internalization of receptor
Fong LG et al. J Biol Chem, 274, 36088-36816 (1999)

signal sequence of cytoplasmic domain
KLKSFK
 cell adhesion
Kosswig N et al. J Biol Chem, 278, 34219-34225 (2003)

signal sequence of cytoplasmic domain
VXFD (rabbit)
internalization of receptor,  transference to cell surface
Morimoto K et al.Biol Pharm Bull, 22, 1022-1026 (1999)
signal sequence of cytoplasmic domain
Ser48 (human)
expression and internalization of receptor, cell migration
Heider H et al. FEBS Letters, 505,  185-190 (2001)
phosphorylation of cytoplasmic domain
Di-leucine motif ?
signal sequence of cytoplasmic domain
 
1. The role of Di-leucine motif in the cytoplasmic     domain of SRA
(1) SR-A mutants/EGFP constructs:
Wild type
N3132LM
EGFP
SRA
SRA
(2) Expression patterns of SRA mutant:
*P<0.05,n=3
EGFP
SR-A            N3132LM
EGFP
SR-A
N3132LM
*
0
200
400
600
800
1000
control
EGFP
SR-A
N3132LM
(3) The internalization of DiI-AcLDL via SRA mutant:

DiI-acLDL
SR-A expression
*
*
DiI-acLDL
*P<0.05,n=3
* *P<0.05,n=3
0
10
20
30
40
EGFP
Wild type
N3132LM
*
EGFP
Wild type
N3132LM
0.02
0.04
0.06
0.08
0.10
0.12
(4) Cell adhesion via SRA mutant:
*
*P<0.05, n=3
*
EGFP
SR-A
N3132LM
Immunofluorescence: The CHO cells were transfected with SRA/EGFP or SRA Mutant/EGFP,
(5) Di-leucine motif  and Clathrin-coated pit:
GFP
Anti-clathrin
overlay
180KD
97KD
     a             b              c
a: EGFP-transfected cell
b: SR-A-transfected cell
c: N3132LM mutant-transfected cell
Clathrin IP:
NΔ1-27
NΔ1-27LM
*
*
*P<0.05,n=3
SR-A     NΔ1-27   NΔ1-27LM
97KD
A
B
C
(6) Expressing of the SRA mutants in CHO cell
EGFP
SR-A
400
600
800
1000
NΔ1-27
NΔ1-27LM
200
(7) The internalization of DiI-AcLDL via SRA mutant:

*P<0.05,n=3
*P<0.05,n=3
*
*
*
*
DiI-acLDL
DiI-acLDL
SR-A expression
10
20
30
40
EGFP
SR-A
NΔ1-27
NΔ1-27LM
0.12
0.10
0.08
0.06
0.04
0.02
0.00
0
EGFP
SR-A
NΔ1-27
NΔ1-27LM
* Di-leucine signal motif in the cytoplasmic domain is required for MSR-A-mediated internalization.


* Di-leucine signal motif-related endocytosis of Ac-LDL may depend on the clathrin-coat pit.

*  There might be multiple signal motives in cytoplasmic domain of MSR, which adapts the multiple functions of MSR. Di-Leucine motif may be an distinct motif from the VXFD in SR-A mediated internalization and adhesion.

2. Identification of peptide ligands for the cytoplasmic domain of SRA
(1) GST -cSRA fusion protein expression and purification:
GST fusion protein were separated on a 12% SDS-PAGE. 1, purified GST fusion protein; 2, E. coli supernatant containing GST fusion protein; 3 and 6, E. coli supernatant; 4, molecular weight standards; 5, E. coli supernatant containing GST.

1      2      3      4      5       6
Western-blot of the fusion protein with
anti-GST antibody. 1, GST-cytoplasmic domain
of SRA fusion protein;2, GST; 3, E. coli supernatant;
4, molecular weight standards.
1          2            3           4  
(2) Screening the phage peptide library with the fusion protein :
The pool of phage becomes enriched in favour of sequences that bind to the target after four
rounds of bio-panning
* phage form unit
** output phages/input phages
Binding specificity of the positive phage clones analysed by ELISA using anti-M13 antibody. The black, binding with the fusion protein (P); The white, binding with GST (N). The positive clones : P/N>3.
(3) Isolation of binding phage clones by  Phage ELISA:
II4        II5       II6        II7       II9      II10      II11     II12      M13
(4) Sequencing the selected positive clones:
* H11: TDRLFMNSIWPG (4, 6, 7, 10, 11, 12)

* H5:  TFWDDGWLLLPR (5)

* H9:  YGDRWFMPVMRS (9)
(5) Searching the PIR-PSD database :
H5: Casein kinase II phosphorylation site (TFWD)

H11:Protein kinase C phosphorylation site (TDR)

(6) Construction of expression plasmids
PEGFP-N1-H5

PEGFP-N1-H9

PEGFP-N1-H11
EGFP
P


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