Cation-independent mannose 6-phosphate receptor: a composite of distinct phosphomannosyl binding sites.
The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR), which contains multiple mannose 6-phosphate (Man-6-P) binding sites that map to domains 3, 5, and 9 within its 15-domain extracytoplasmic region, functions as an efficient carrier of Man-6-P-containing lysosomal enzymes. To determine the types of phosphorylated N-glycans recognized by each of the three carbohydrate binding sites of the CI-MPR, a phosphorylated glycan microarray was probed with truncated forms of the CI-MPR. Surface plasmon resonance analyses using lysosomal enzymes with defined N-glycans were performed to evaluate whether multiple domains are needed to form a stable, high affinity carbohydrate binding pocket. Like domain 3, adjacent domains increase the affinity of domain 5 for phosphomannosyl residues, with domain 5 exhibiting approximately 60-fold higher affinity for lysosomal enzymes containing the phosphodiester Man-P-GlcNAc when in the context of a construct encoding domains 5-9. In contrast, domain 9 does not require additional domains for high affinity binding. The three sites differ in their glycan specificity, with only domain 5 being capable of recognizing Man-P-GlcNAc. In addition, domain 9, unlike domains 1-3, interacts with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) oligosaccharides containing a single phosphomonoester. Together, these data indicate that the assembly of three unique carbohydrate binding sites allows the CI-MPR to interact with the structurally diverse phosphorylated N-glycans it encounters on newly synthesized lysosomal enzymes.
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Richard N
RN
Bohnsack
Xuezheng
X
Song
Linda J
LJ
Olson
Mariko
M
Kudo
Russell R
RR
Gotschall
William M
WM
Canfield
Richard D
RD
Cummings
David F
DF
Smith
Nancy M
NM
Dahms
D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008360:Mannosephosphates / Q000737:chemistry / Q000378:metabolism*; D046228:Microarray Analysis; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011134:Polysaccharides / Q000032:analysis; D017527:Receptor, IGF Type 2 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D008360:Mannosephosphates; D011134:Polysaccharides; D017527:Receptor, IGF Type 2; C027693:mannose-6-phosphate; D005966:Glucuronidase