Interactions between bovine seminal plasma proteins and model

lipid membranes 

Danny Lassiseraye1, Puttaswamy Manjunath2 and Michel Lafleur1

 

 1Département de chimie, Université de Montréal

2Centre de recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont

Introduction

The bovine seminal plasma contains a family of proteins (BSP-A1/A2, BSP-A3 and BSP-30kDa, collectively called « BSP ») that bind to sperm membrane upon ejaculation. It has been shown that these proteins have a particular affinity for lipids bearing the choline moiety. The aim of our studies is to understand how these proteins interact with lipids. We examined the interactions between BSP proteins and lipid vesicles of different compositions. Isothermal titration calorimetry (ITC) has been used to determine the partition coefficients of the proteins with vesicles containing neutral lipids, charged lipids, and cholesterol. This technique also provided informations on the molar binding enthalpy, on the number of lipids interacting with one protein and on the effective charge of BSP upon binding. The effect of the BSP proteins on the morphology of the vesicles has been investigated with cryo-EM, showing a curvature effect on the vesicles and the formation of small vesicles. Finally, infrared spectroscopy indicated that the structure of the BSP proteins is stabilized by the presence of lipids.

Isothermal Titration Calorimetry

ITC can measure « in situ » the enthalpy (H) variation associated to a ligand binding to its substrate.

BSP proteins are injected in the sample cell containing lipid vesicles. The binding enthalpy is detected by the variation of electrical power needed to maintain both cells at the same temperature.

Temperature effect on the binding of BSP proteins to POPC vesicles

-0.2

0.0

0.2

0.40 100 200 300

Time (min)

Hea

t flo

w (µ

cal/s

ec)

0.000 0.002 0.004 0.006 0.008 0.010

0

60

120

Protein-to-lipid molar ratio

Tot

al e

ntha

lpy

(kJ/

mol

of i

njec

tant

)

10°C 20°C 37°C

Temperature effect on the binding of BSP proteins to POPC/POPG vesicles

-2

-1

0

0 100 200 300

Time (min)H

eat

flow

(µ

cal/s

ec)

0.000 0.002 0.004 0.006 0.008 0.010-1200

-1000

-800

-600

-400

-200

Protein-to-lipid molar ratio

Tot

al e

ntha

lpy

(kJ/

mol

of i

njec

tant

)

20°C 37°C

Temperature effect on the binding of BSP proteins to POPC/Chol vesicles

0.0

0.5

1.0

1.5

0 50 100 150

Time (min)H

ea

t flo

w (

µca

l/se

c)

0.000 0.002 0.004 0.006 0.008 0.010

0

50

100

150

200

250

300

Protein-to-lipid molar ratio

Tot

al e

ntha

lpy

(kJ/

mol

of in

ject

ant)

20°C 37°C

ITC Results

Table 1 : Membrane saturation (lipids/BSP) and molar binding enthalpy for the titration of four lipid systems with BSP proteins. Saturation ratios are calculated taking into account only the outer leaflet of the lipid vesicles.

Table 2 : Partition coefficients and effective charge for the binding of BSP proteins to the four studied systems.

a : Value only attributed to the transfer of the protein in the lipid membrane.

b : Value including the electrostatic interactions and the transfer of the protein in the lipid membrane

Conclusions - ITC• Affinity : PC/PE > PC/PG > PC > PC/Chol.

• Temperature affects the titration profiles. – A multimer-dimer transition was reported for the BSP proteins at 36°C.– However, considerable differences were observed as a function of membrane

composition.– Thus, the resulting behavior appears to reflect both the solution structure of

BSP proteins and variation of membrane composition.

• The saturation molar binding ratio (Lipid/BSP) changes with the lipid composition of vesicles.

• The presence of a negative charge on the membrane leads to higher molar binding enthalpy (H), suggesting electrostatic interactions.

Infrared Spectroscopy

1700 1680 1660 1640 1620 1600

Wavenumber (cm-1)

20 °C

60 °C Spectra series of free BSP in solution, showing the evolution of the Amide I band between 20 and 60°C.

The maximum of the band is shifted towards higher frequencies.

Amide I band shift of free and lipid-bound BSP

The presence of lipids affects the temperature at wich the maximum of the Amide I band is shifted.

The binding to lipids seems to stabilize the protein structure.

Cryo-EM

Addition of BSP (Ri 48) to POPC LUV

POPC unilamellar vesicles

Cryo-EM

Addition of BSP (Ri 48) to PC/Chol LUV

Addition of BSP (Ri 48) to PC/PE LUV

Conclusions – Cryo-EM

• Addition of BSP modifies the morphology of the vesicles.

• Formation of tube-like structures, of stretched and connected structures, and budding.

• Tube-like structures seem to have a smaller diameter with the PC/PE system.

• Stretched and connected structures seem to be more abundant with the PC/Chol system.

General Conclusions

• The lipid/BSP saturation ratio correlates with the order of the bilayer: PC/Chol > PC/PE > PC ≈ PC/PG.

• This may be related to the morphology changes of the vesicles upon BSP proteins binding.

• The binding to lipid membranes stabilizes the BSP proteins solution structure.

• Temperature affects the binding of BSP proteins to vesicles of different lipid compositions.