High-level expression and characterization of a secreted recombinant cation-dependent mannose 6-phosphate receptor in Pichia pastoris

Abstract

Mannose 6-phosphate receptors (MPRs) form essential components of the lysosomal enzyme targeting system by binding newly synthesized acid hydrolases with high (nM) affinity. We report the use of Pichia pastoris as a host to efficiently express the extracytoplasmic ligand-binding domain of the cation-dependent mannose 6-phosphate receptor. A truncated and glycosylation-deficient form of the receptor AF-Asn 81/Stop 155 was secreted into the culture medium, yielding ∼28 mg/L after purification, which is an improvement of 10–100-fold compared to expression in baculovirus-infected insect cells and mammalian cells, respectively. Enzymatic deglycosylation indicated high-mannose sugars at the single potential glycosylation site of Asn 81. The extent and heterogeneity of N-glycans were revealed by applying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In the case of AF-Asn 81/Stop 155, the majority (75%) of the oligosaccharides contained chain lengths of Man 8–10GlcNAc 2 while Man 11–12GlcNAc 2 comprised the remaining (25%) N-linked sugars. A comparative MALDI-TOF spectra of Asn 81/Stop 155 purified from insect cells indicated that Man 2–3GlcNAc 2 and GlcNAcMan 2–3GlcNAc 2 share the oligosaccharide pool. The receptor isolated from yeast was functional with respect to ligand binding and acid-dependent dissociation properties, as determined by pentamannosyl phosphate-agarose affinity chromatography. In addition, the protein was biochemically and functionally similar to Asn 81/Stop 155 expressed in insect cells concerning its oligomeric state and binding affinity to the lysosomal enzyme, β-glucuronidase ( K d=1.4 nM). These results demonstrate that P. pastoris is a convenient system for the production of large quantities of functional recombinant MPRs suitable for structure–function studies.