Elucidating the role of Cosmc in the regulation of T-synthase biosynthesis Público

Abstract

Elucidating the Role of Cosmc in the Regulation of T-synthase Biosynthesis

Cosmc (Core 1 β3-galactosyltransferase Specific Molecular Chaperone) plays an
important role in the regulation of O-glycan biosynthesis for animal cell glycoproteins. T-
synthase (Core 1 β3-galactosyltransferase) is the key Golgi residing enzyme that initiates
the formation of extended O-glycans by catalyzing the addition of galactose to the Tn
antigen (GalNAcα1-Ser/Thr) to form the core 1 O-glycans (Galβ1-3GalNAcα1-Ser/Thr).
The core 1 O-glycan is the precursor structure for many extended O-glycans. Lack of
Cosmc function in vivo results in an inactive oligomeric complex of T-synthase that is
eventually degraded, leading to loss of T-synthase activity and expression of aberrant O-
glycans. Although Cosmc is required for the formation of catalytically active T-synthase
in vivo, its mechanism is largely unknown. We developed an in vitro functional assay and
showed that Cosmc specifically promotes the activity of partially denatured T-synthase
independently of other factors, and that Cosmc does not bind native T-synthase.
Furthermore, we generated active Cosmc conjugated beads, and demonstrated that Cosmc
directly interacts with only non-native T-synthase to form a relatively stable noncovalent
tight complex. Unexpectedly, the T-synthase within the complex is reactivated. ATP did
not dissociate this complex suggesting that it does not regulate the Cosmc chaperone
cycle. Furthermore, Cosmc interaction and release of reactivated T-synthase is not
regulated by redox, calcium and pH. The primary factor that shifted the equilibrium
between Cosmc and reactivated T-synthase was excess denatured T-synthase itself. These
studies, along with others, suggest that newly synthesized T-synthase interacts with
Cosmc in the ER to promote its folding in that compartment. This process leads to the
formation of a transient complex between Cosmc and a catalytically active T-synthase.
The T-synthase within Cosmc complex is released in the presence of more newly
synthesized T-synthase, which interacts with Cosmc for another round of refolding.
These findings have significantly contributed in our understanding of the molecular
mechanism of Cosmc function, which may in turn lead to further understanding of
protein O-glycan biosynthesis and several different diseases, such as Tn syndrome, IgA
nephropathy, Henoch-Schönlein purpura, and malignant transformation.

Table of Contents

CHAPTER 1: INTRODUCTION
1.1 Protein folding 1
1.2 In vitro verses in vivo protein folding 2
1.3 Assisted protein folding 3
1.3.1 Molecular chaperone 4
1.3.1.1 General Chaperones 4
Hsp70 Chaperone 5
Hsp90 6
Small heat shock proteins (sHsp) 6
Chaperonins 7
1.3.1.2 Specific molecular chaperones 7
1.4. A brief summary of information known about T-synthase and its specific
chaperone Cosmc prior to this dissertation 11
T-synthase 11
Cosmc 12
1.5 Protein folding in the ER 13
1.5.1 Unfolded Protein response 14
1.5.2 Protein glycosylation 15
1.5.3 Glycosyltransferase 16
1.6 Mucin type O-glycosylation 18
1.6.1 Mucin structure 18

1.6.2 Biosynthesis Mucin type O-glycosylation 21
1.7 Significance of Cosmc in relation to human diseases 23
1.7.1 Tn syndrome 23
1.7.2 Cancer 23
1.7.3 IgA nephropathy and Henoch Schönlein Purpura 24
1.8 Focus of this dissertation 25
1.9 References 27

CHAPTER 2: The Endoplasmic Reticulum Chaperone Cosmc directly promotes
in vitro folding of T-synthase 38
2.1 Abstract 39
2.2 Introduction 39
2.3 Experimental Procedure 41
Materials 41
Preparation of Expression Construct 41
Expression and purification of 6xHis-sCosmc, 6xHis-smCosmc,
and soluble N-terminus HPC4-tagged Core 1 β3-Gal-T (T-synthase) 42
In vitro reconstitution of heat-denatured T-synthase and heat-denatured
β4-GalT 42
Chemical denaturation and renaturation of T-synthase 43
Luciferase renaturation assay 43
Time dependency for restoration of HpC4-sT-syn 44
2.4 Results 44
2.4.1 Cosmc promotes renaturation of denatured T-synthase in vitro 44
2.4.2 Cosmc promotes renaturation of heat-denatured T-synthase in a dose

dependent manner 50

2.4.3 Mutated form of Cosmc does not restore the activity of heat-denatured

T-synthase as efficiently as soluble Cosmc 53

2.4.4 Cosmc does not require ATP for its in vitro renaturation of heat-denatured

T-synthase 56

2.4.5 Cosmc cannot restore the activity of denatured β1-4-Galactosyltransferase

(β4-GalT) 59

2.5 Discussion 62

2.6 References 67

CHAPTER 3: Cosmc directly facilitates folding of T-synthase to promote

correct protein O-glycosylation 72

3.1 Abstract 73

3.2 Introduction 74

3.3 Materials and Methods 76

Preparation of different versions of Cosmc and T-synthase 76

Preparation of anti-HPC4 and Cosmc conjugated beads 77

Characterization of recombinant HPC4-sT-synthase using

Asialo-BSM beads 77

In vitro reconstitution experiments 78

In vitro reconstitution experiments with Cosmc beads and

pull down experiments 78

N-Ethylmaleimide (NEM) reaction 79

Sodium Arsenite reaction 79

Release experiments 79

Quantification of Western blot data 80

Plasmid Construction 80
Recombinant Protein Expression and Purification 81
In-solution refolding followed by pull down experiments 81
Treatment of Cosmc by 6 M GnHCl 81
Crosslinking 82
Chromatography 82
3.4 Results 82
3.4.1 Cosmc does not promote the activity of native T-synthase but
promotes the refolding of denatured T-synthase 82
3.4.2 Cosmc forms a stable complex with non-native but not with
native T-synthase 89
3.4.3 Cosmc associates with reconstituted active T-synthase but not
with native T-synthase 93
3.4.4 ATP does not cause release of reconstituted active T-synthase
associated with Cosmc 100
3.4.5 Cosmc folds T-synthase independently of intermolecular disulfide
bond formation 105
3.4.6 Denatured T-synthase is sufficient for the release of reconstituted
active T-synthase bound to Cosmc 111
3.5 Discussion 115
3.6 References 122

CHAPTER 4: Discussion and Future direction 126
4.1 Cosmc refolds T-synthase from unfolded state in vitro 126
4.2 Cosmc is a bona fide chaperone for T-synthase 127
4.3 Chaperone cycle of Cosmc 129

4.4 Future Studies 133
4.5 References 138

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Cell
Palavra-chave	O-glycosylation ER quality Control Specific molecular chaperone Cosmc T-synthase
Committee Chair / Thesis Advisor	Cummings, Richard, Emory University
Committee Members	Wilkinson, Keith D, Emory University Matsumura, Ichiro, Emory University Cheng, Xiaodong, Emory University Fu, Haian, Emory University

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