Localization of the Carbohydrate Recognition Sites of the Insulin-like Growth Factor II/Mannose 6-Phosphate Receptor to Domains 3 and 9 of the Extracytoplasmic Region

Abstract

The insulin-like growth factor II/mannose 6-phosphate receptor is a multifunctional receptor that binds to a diverse array of mannose 6-phosphate (Man-6-P) modified proteins as well as nonglycosylated ligands. Previous studies have mapped its two Man-6-P binding sites to a minimum of three domains, 1-3 and 7-9, within its 15-domain extracytoplasmic region. Since the primary amino acid determinants of carbohydrate recognition by the insulin-like growth factor II/mannose 6-phosphate receptor are predicted by sequence alignment to the cation-dependent mannose 6-phosphate receptor to reside within domains 3 and 9, constructs encoding either domain 3 alone or domain 9 alone were expressed in a Pichia pastoris expression system and tested for their ability to bind several carbohydrate ligands, including Man-6-P, pentamannosyl phosphate, the lysosomal enzyme, ␤-glucuronidase, and the carbohydrate modifications (mannose 6-sulfate and Man-6-P methyl ester) found on Dictyostelium discoideum lysosomal enzymes. Although both constructs were functional in ligand binding and dissociation, these studies demonstrate the ability of domain 9 alone to fold into a high affinity (K d ‫؍‬ 0.3 ؎ 0.1 nM) carbohydrate-recognition domain whereas the domain 3 alone construct is capable of only low affinity binding (K d ϳ 500 nM) toward ␤-glucuronidase, suggesting that residues in adjacent domains (domains 1 and/or 2) are important, either directly or indirectly, for optimal binding by domain 3.

Identification of a Low Affinity Mannose 6-Phosphate-binding Site in Domain 5 of the Cation-independent Mannose 6-Phosphate Receptor

“…3, inset, lanes 2-4) is consistent with the singly glycosylated form of Dom5His and indicates that the two potential N-glycosylation sites in Dom5His are utilized. Interestingly, as we have observed with other MPR constructs expressed in P. pastoris (19,34,40), the culture conditions influence the extent of N-glycosylation: Dom5His purified from a 50-ml culture did not reveal any singly glycosylated species (Fig. 3, inset, lane 1), whereas that purified from large cultures (Ն500 ml) was more heterogeneous, containing both the singly and doubly glycosylated forms in comparable amounts (data not shown).…”

“…Briefly, the sequences encoding domain 5 (residues 584 -725) followed by six histidine residues (CAC) and a stop codon (TGA) were amplified by PCR and subcloned into the P. pastoris expression vector pGAPZ␣A, which utilizes a constitutive promoter, in-frame with the Saccharomyces cerevisiae ␣-factor signal sequence. The Dom9His construct (residues 1184 -1327 followed by a His 6 tag) was subcloned into the pGAPZ␣A vector as described previously (19). DNA sequencing (Protein and Nucleic Acid Core Facility, Medical College of Wisconsin) confirmed the predicted sequences.…”

“…Man-6-P Binding Property of Dom5His-Our initial attempts to assess phosphomannosyl binding using iodinated ␤-glucuronidase, an assay that can readily detect interactions in the low micromolar range (19), failed to detect specific binding to Dom5His up to a ligand concentration of 250 nM (data not shown). To further explore the possibility that Dom5His binds Man-6-P or other carbohydrates, we took a more sensitive …”

“…Sequence analysis of each of the 15 domains of the CI-MPR demonstrated significant amino acid identity to each other (16 -38%) and to the ϳ150-residue extracytoplasmic region of the CD-MPR (14 -28%) (15). In contrast to the CD-MPR, which binds one molecule of Man-6-P/polypeptide (16), the CI-MPR contains two high affinity (nanomolar) Man-6-P-binding sites, which have been mapped to domains 1-3 and 9 (17)(18)(19), with essential residues localized to domains 3 and 9 (20,21). The CI-MPR, unlike the CD-MPR, also interacts with a number of non-Man-6-P-containing molecules that include insulin-like growth fac-tor II, plasminogen, the urokinase-type plasminogen activator receptor, and retinoic acid.…”

Binding of Urokinase-type Plasminogen Activator Receptor (uPAR) to the Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor

“…Marron-Terada et al (50) showed that truncated Man-6-P/ IGF2R receptors encompassing repeats 1-3 or repeats 7-9 displayed similar affinities for Man-6-P, but the two Man-6-P binding domains differed in the binding activity under acidic conditions and in their preferences for binding various lysosomal enzymes from Dictyostelium discoideum, which contain modified Man-6-P residues. Additionally, Hancock et al (11) demonstrated that constructs encoding repeat 3 or 9 alone expressed in Pichia pastoris were each able to bind several Man-6-P ligands, but the repeat 9 receptor alone could bind the ligands with high affinity. The resin pellets were collected by centrifugation, washed, and incubated with aliquots (ϳ500 g of protein) of HT-1080 plasma membrane Triton X-100 extracts in a final volume of 0.5 ml.…”