Selective endocytosis of recombinant human BMPs through cell surface heparan sulfate proteoglycans in CHO cells: BMP-2 and BMP-7

Mi Gyeom Kim,Gyun Min Lee,Young Sik Kim,Che Lin Kim,Ju Woong Jang

doi:10.1038/s41598-021-82955-1

Abstract

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis results in poor yields of recombinant human bone morphogenetic proteins (rhBMPs) from CHO cell cultures. Upon incubation of rhBMP-2 and rhBMP-7 with CHO cells at 37 °C, both rhBMP-2 and rhBMP-7 bound to the cell surface HSPGs in CHO cells, but only rhBMP-2 was actively internalized into CHO cells. Cell surface HSPGs were found to serve as the main receptor for rhBMP-2 internalization. It was also found that the cell surface HSPG-mediated endocytosis of rhBMP-2 occurred through both the clathrin- and caveolin-dependent pathways. Blockage of rhBMP-2 internalization by the addition of structural analogs of HSPGs such as dextran sulfate (DS) and heparin dramatically increased rhBMP-2 production in recombinant CHO (rCHO) cell cultures. Compared to the control cultures, addition of DS (1.0 g/L) and heparin (0.2 g/L) resulted in a 22.0- and 19.0-fold increase in the maximum rhBMP-2 concentration, respectively. In contrast, the production of rhBMP-7, which was not internalized into the rCHO cells, did not dramatically increase upon addition of DS and heparin. Taken together, rhBMPs have a different fate in terms of HSPG-mediated internalization in CHO cells. HSPG-mediated endocytosis of each rhBMP should be understood individually to increase the rhBMP yield in rCHO cell cultures.

Highlights

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis results in poor yields of recombinant human bone morphogenetic proteins from CHO cell cultures
We found that only recombinant human bone morphogenetic proteins (rhBMPs)-2 was internalized into CHO cells via cell surface HSPG binding
To study whether extracellular recombinant human BMP-2 (rhBMP-2) and rhBMP-7 are internalized via cell surface HSPGs into CHO cells, medium containing 10 μg/ mL of rhBMP-2 or rhBMP-7 was incubated at 4 °C and 37 °C with and without DG44 cells in the absence or presence of 0.5 g/L dextran sulfate (DS) or 0.1 g/L heparin

Summary

Introduction

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis results in poor yields of recombinant human bone morphogenetic proteins (rhBMPs) from CHO cell cultures. Blockage of rhBMP-2 internalization by the addition of structural analogs of HSPGs such as dextran sulfate (DS) and heparin dramatically increased rhBMP-2 production in recombinant CHO (rCHO) cell cultures. Endocytosis of secreted rhBMP-4 through cell surface heparan sulfate proteoglycans (HSPGs), which served as the major receptors, results in poor yields of rhBMP-4 from CHO cell c ultures[8]. DS was added to the culture medium to increase the rhBMP-2 production, but the mechanism by which DS mediates enhanced rhBMP-2 production has not been understood y et[10] It has never been examined before whether endocytosis of secreted rhBMP-2 and rhBMP-7 through cell surface-bound HSPGs occurs during CHO cell cultures. The functional roles and HS binding may differ between rhBMP-2 and rhBMP-7 due to structural differences

Methods

Results

Conclusion