Protein sorting by high performance liquid chromatography. 2. Separation of isophosphorylates of recombinant human DNase I on a polyethyleneimine column

Abstract

Anion exchange HPLC with a polyethylenimine (PEI) column separates recombinant human deoxyribonuclease I (rhDNase) glycoforms according to the extent and positions of phosphorylation of mannose residues in N-linked oligosaccharides. The separation provides a selectivity unavailable by anion exchange HPLC with other columns or by isoelectric focusing gel electrophoresis and can be used to quantify the phosphate content of preparations of rhDNase. Tryptic mapping of fractions collected from the column and treated with alkaline phosphatase was used to identify the sites of phosphorylation. Unnatural forms of rhDNase, bearing oligosaccharide structures at only one of the two sites of glycosylation, were prepared by cleaving the phosphate-containing high mannose and hybrid structures from the purified isophosphorylates fractionated on the PEI column. The separation of rhDNase isophosphorylates on the PEI column mimics the relative affinities for the mannose 6-phosphate receptor that traffics acid hydrolases to lysosomes and provides a useful example of protein sorting by biomimetic interaction chromatography.

Susceptibility of rhDNase I to Glycation in the Dry-Powder State

“…In the crystal structure of bovine DNase, the asparagine 18-linked carbohydrate structure is unresolved past the first two sugar residues due to its flexibility; 10 however, the bovine carbohydrate structure is not phosphorylated. 8 Therefore, the orientation of the rhDNase phosphorylated carbohydrate structure has not been determined; however, no other acidic negative charges are near the lysine 50 positive surface charge which could repel potential interactions with the negatively charged phosphorylated mannose residues. Lysine 77 resides on an exposed loop in the active site and is readily reactive with lactose, with four nearby arginine guanidino groups.…”

“…This multiply charged, acidic protein is wellcharacterized by many of the standard techniques for compositional and sequential analyses, as well as some uniquely adapted chromatographic separations for the intact protein. 7,8,9 This report describes the characterization of an experimental dry-powder form of rhDNase, formulated with the reducing disaccharide lactose for use in a new, dry-powder inhalation device that is more convenient for patient usage. The results demonstrate that reaction occurs in the dried powder formulation between the reducing sugar lactose and several accessible amino groups in the protein molecule, causing the formation of a covalently modified, lactosated (glycated by lactose) protein variant.…”

“…The protein is glycosylated at both consensus sites for asparagine linkage with complex, hybrid, and high-mannose carbohydrate structures, which possess varying amounts of sialylation and phosphorylation. This multiply charged, acidic protein is wellcharacterized by many of the standard techniques for compositional and sequential analyses, as well as some uniquely adapted chromatographic separations for the intact protein. ,, …”

Human DNase I Contains Mannose 6-Phosphate and Binds the Cation-Independent Mannose 6-Phosphate Receptor

“…Ion exchange HPLC on a PEI column, shown in Figure 3, provides a convenient method for quantitative estimation of the extent of phosphorylation of hDNase samples, as well as a means of preparing isophosphorylates for further characterization (70). hDNase derived from urine can be separated by this column into four major peaks that differ in the extent of phosphorylation of the enzyme.…”

“…Hence, anion exchange HPLC on the PEI column is selective for phosphate residues on the protein. The four peaks in the chromatogram of rhDNase contain either zero (peak I), one (peaks II and III), or two (peak IV) phosphate monoesters (70). Peaks II and III differ in the location of the monophosphorylated oligosaccharide at either Asn-106 or Asn-18, respectively.…”