Studies on the Substrate Specificity of Neutral α-Mannosidase Purified from Japanese Quail Oviduct by Using Sugar Chains from Glycoproteins

Abstract

The substrate specificity of neutral α-mannosidase purified from Japanese quail oviduct [Oku, H., Hase, S., & Ikenaka, T. (1991) J. Biochem. 110, 29-34] was analyzed by using 21 oligomannose-type sugar chains. The enzyme activated with Co²⁺ hydrolyzed the Manα1-3 and Manα1-6 bonds from the non-reducing termini of Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβl-4GlcNAc (M5A), but hardly hydrolyzed the Manα1-2 bonds of Man₉GlcNAc₂. The hydrolysis rate decreased as the reducing end of substrates became more bulky: the hydrolysis rate for the pyridylamino (PA) derivative of M5A as to that of M5A was 0.8; the values for M5A-Asn and Taka-amylase A having a M5A sugar chain being 0.5 and 0.04, respectively. The end product was Manβ1-4G1cNAc₂. For the substrates with the GlcNAc structure at their reducing ends (Man₅GlcNAc, Man₆G1cNAc and Man₉GlcNAc), the hydrolysis rate was remarkably increased: Man₅GlcNAc was hydrolyzed 16 times faster than M5A, and Man₉GlcNAc 40 times faster than Man₉GlcNAc₂. The enzyme did not hydrolyze Manα1-2 residue(s) linked to Manα1-3Manβ1-4GIcNAc. The end products were as follows:
Manα1
6 Manβ1-4GlcNAc
3
(Manα1-2)_{0_??_2}Manα1
These results suggest that oligomannose-type sugar chains with the G1cNAc structure at their reducing ends seem to be native substrates for neutral α-mannosidase and the enzyme seems to hydrolyze endo-β-N-acetylglucosaminidase digests of oligomannose-type sugar chains in the cytosol.