Background: a bit about…Background: a bit about…

Important roles in:

•Glycoprotein biosynthesis, quality control & catabolism

•Multiple forms of α-mannosidases in mammalian cells, differing in specificity, function and cellular location

Classified into 2 groups conserved through eukaryotic evolution

Class I

- Narrow specificity for α-1, 2-mannosidic linkages

-Further specificity within family for isomeric species produced

Class II

- Broad specificity: hydrolyse α(1→ 2), α(1→3) and α(1→6) mannosidic linkages

Manß1-4GlcNAcß1-4GlcNAcß1-Asn

Man 1-6

Man1-3

Man1-6

Man1-3

Man1-2

Man1-2

Man1-2Man 1-2

ERAD

misfolded

M8B

/ M8C

persistently misfolded

Monosaccharide Monosaccharide constituents

ERER

LysosomeLysosome

CytosolCytosolCELL

G1-3M8N1

G1-3M5N1

G1-3M4N1

?

G1-3M4N1

lysosome

Plasma membrane

Chitobiase

ProteasomeM8N1

M5N1

Chitobiase

Cytosolicα-mannosidase

Cytosolicα- mannosidase

Cyt α-man ?

Fast

Slow

Protein and Oligosaccharide Processing in the Cytosol of ERAD

G1-3M8N2 + M8N2+

M4N1

Cyt α-man?

HN

OHHOOH

HON OH

OHHHO

N

NH

HHO

HO

OH

HO

O

O HN

OHHO

H

HO

HO

148 266149 265

N

OHHO

OHHO

N

OHHO

OHHON

OHHO

OH

N

OHHO

OH

Deoxymannojorimycin (DMJ) Kifunensin (Kif) Swainsonine (SW)

1, 4 deoxy-1,4-imino D-mannitol (DIM)

Known α-mannosidase inhibitors

Novel 7-membered imino-sugars: inhibitory effect on cellular α-mannosidases (Kukushkin & Butters; unpublished data)

Weak general inhibitor of mammalian α-mannosidases

Low conc.: Golgi mannosidase I and Man9-mannosidase

Higher conc.: Will start to inhibit other α-mannosidases

Potent and specific inhibitor of Golgi mannosidase I

ER mannosidase I and Man9-mannosidase

Inhibits lysosomal mannosidase and Golgi mannosidase

Project Aims

The aims of my project were:

To define α-mannosidase targets of novel inhibitors in cells and compare these to known α-mannosidase inhibitors

This will be accomplished by:

1.Evaluation of cytosolic and lysosomal α-mannosidase inhibition by measuring free oligosaccharides (FOS) in HL60 and MDBK cell lines.

2.Increasing our knowledge of mannosidases within their cellular pathways in cell lines above.

3.Using glycoprotein precipitation, isolation and PNGase glycan release methods to observe effects of inhibitors on glycan species of total cellular glycoprotein.

4.Identification of lysosomal FOS species following cellular fractionation. Two cell lines will be used that have differing biosynthetic and catabolic pathways: HL60, a human promyelocytic leukaemia cell line and MDBK, a bovine kidney cell line.

5.Estimation of ER and Golgi α-mannosidase inhibition by using Fluorescence-activated cell sorting (FACS) to analysing glycoprotein derived oligosaccharides on the cell surfacee following treatment with α-mannosidase inhibitors.

Flu

ore

scen

ce

(arb

itra

ry u

nit

s) M4N

M5NG1M5 M9N

M7N

M4N

M5N

G1M5 M7N/N2 M8N/N2

M9N/N2

M7N/N2 M8N G1M9N?

M8N/N2M9N/N2

M7N/N2

Untreated HL60

DMJ treated(1mM)

DIM treated (100μM)

HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from HL60 cells treated with known α-mannosidase inhibitors

M5N

M3N2

M4N2M8N2

M5N

M3N2

M5N2 M9N/N2M6N2 M7N/N2

G1M5NM4N2

M4N

M9N/N2

Untreated MDBK cells

DIM treated (100μM)

HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with known α-mannosidase inhibitors

3000

6000

Flu

ore

sc

en

ce

(a

rbit

rary

un

its

)

Minutes

-

-

22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

M8N2

M7N2

M4N2G1M5

M5N

M4N2M5N

G1M5

M8N2

M8N2

M7N2

M9N

M9N

M9N/N2

M9N/N2

M7N2M6N2

HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with Kif at increasing concentrations (1μM- 100μM)

Untreated

+ 1μM Kif

+ 10μM Kif

+ 100μM Kif

Flu

ore

sc

en

ce

(a

rbit

rary

un

its

)

SATIN - Fluorescence

M9N

M5N

M4N

M6N M7N/N2

G1M5

22 24 26 28 30 32 34 36 38 40 42

Minutes

M5N

M5N

M9N

M7N/N2

G1M9

G1M9

G1M5

M6N

G1M5

M8N

M8N

M8N M9N

M4N

M4N

M6N2

M6N2

HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from HL60 cells treated with 148 and 149

Flu

ore

sc

en

ce

(a

rbit

rary

un

its

)

-

M3N2

M4N2

M5N

M7N2

18 20 22 24 26 28 30 32 34 36 38 40 42

M3N2 M4N2

M4N2

M5N

M5N

M6N2

M6N2 M7N2 M8N2

M8N2

6000

1800

1200

M9N/N2

G1M5

M9N/N2

M5N2

M5N2

HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with 148 and 149

Flu

ore

sc

en

ce

(a

rbit

rary

un

its

)

Minutes

HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from HL60 cells treated with 265 and 266

2000

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

1600

4000

Minutes

M5N

M5N

M5N

M4N

M4N

M4N

M3N

M3N

M3NM9N

M9N

M9N

M6N

M6N

Untreated

+ 100μM 265

+ 100μM 266

Flu

ore

sc

en

ce

(a

rbit

rary

un

its

)

HPLC analysis of 2-AA fluorescently labelled free oligosaccharides (FOS) from MDBK cells treated with 265 and 266

SATIN - Fluorescence

6000

0

6000

Minutes12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00

1300

M3N2

M3N2

M3N2

Flu

ore

sc

en

ce

(a

rbit

rary

un

its

)

Effect on total cellular glycoprotein upon inhibitor treatment

Elution time (minutes)

600

1000

SATIN - Fluorescence

1200

1000

1000

SATIN - Fluorescence

2500

22 24 26 28 30 32 34 36 38 40 42 44

M9N2

flu

ore

sc

en

ce

% increase in ConA binding after 24hrs

% increase in ConA binding after 72hrs

+ 100μM Kif 68.1% 136%

+ 100μM 148 13.9% 34.2%

Flow cytometry data representing % change in ConA binding in Kif and 148 treated HL60 cells over 24hrs and 72 hrs

Untreated HL60

+ DMJ (1mM)

+ 148 (100μM)

+ 265 (100μM)

+ 266 (100μM)

+ Kif (100μM)

Cellular fractionation of untreated HL60 and MDBK cells

HL60 Cells Lysosomal Activity

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0 2 4 6 8 10 12 14

Cell Fraction Number

Lys

oso

mal

Act

ivit

y/ u

g p

rote

in Fraction 9

42

Flu

ore

scen

ce

(arb

itrar

y un

its)

16 18 20 22 24 26 28 30 32 34 36 38 40

Elution time (minutes)

MDBK Cells Lysosomal Activity

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14

Fraction number

lyso

som

al a

ctiv

ity/ u

g pr

otei

n

Fraction 8

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42

In SummaryKnown α-mannosidase inhibitors:

- DMJ: low potency, low specificity α-mannosidase inhibitor. Does not inhibit cytosolic mannosidase at high concentration (1mM) but most likely inhibiting ER/Golgi and partially inhibiting lysosomal α-mannosidases.

- Kif: Known strong inhibitor of ER Mannosidase I and Golgi mannosidases. No inhibition of ER Man II due to accumulation of M8C.

- Very high potency, inhibitory effects observed from 1μM conc. No inhibition of cytosolic/lysosomal α-mannosidases. Selective for ER/Golgi mannosidases and the biosynthetic pathway for glycoprotein maturation.

- DIM: Selective for lysosomal α-mannosidase and lysosomal α1,6 mannosidase. Partial inhibition of catabolism of high mannose structures, strong inhibition of breakdown of core N-linked glycan isomer M3aN2.

Novel α-mannosidase inhibitors

- 148 & 149: Very similar inhibitory patterns in the cell. Inhibits cytosolic α-mannosidase, and lysosomal mannosidase in a similar way to DIM. Subtle inhibition of conversion of oligomannose to complex glycan maturation to the cell surface.

- 265: No inhibition of cytosolic α-mannosidase. Targets lysosomal α-mannosidase and lysosomal α1, 6 mannosidase with particularly potent inhibition for the catabolism of core M3aN2.

- 266: No inhibition of cytosolic α-mannosidase. Inhibits lysosomal α-mannosidase with particularly potent inhibition for the catabolism of core M3aN2. Partial inhibition of catabolism of high oligomannose glycans by lysosomal α-mannosidase . Low levels of inhibition for α1,6 mannosidase.

Future work• Cellular fractionation with added inhibitor followed by FOS isolation and characterisation to

confirm and clarify target compartments of novel inhibitors within the cell and to specify enzymes inhibited.

• Carry out Flow Cytometry experiment with 266 and 265 to analyse effect on glycoprotein maturation at the cell surface

• To confirm the cytosolic inhibition of 148 and 149. Proposed experiment: Addition of 148/149 following treatment of cells with glucosidase inhibitor such as NB-DMJ to create more tri-glucosylated species. If 148/149 inhibit in the cytosol, will generate more glucosylated high-mannose structures from FOS analysis.

• May want to develop a more selective or & more potent inhibitor of the cytosolic α-mannosidase: drug screening with a range of 148,149-like structures.

Acknowledgements

Dr Terry Butters

Dr David Neville

Dr Dominic Alonzi

Stephanie Boomkamp

Professor Raymond Dwek