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Human lysosomal arylsulfatase A (ASA) is a member of the highly conserved
sulfatase gene family. It is synthesized as a 507 amino acid precursor
and is processed in the endoplasmic reticulum to yield a mature 489 amino
acid protein with a calculated mass of 51,908 Da. ASA's major natural substrate
is cerebroside 3-sulfate which will accumulate if there is a deficiency
in ASA, resulting in a lysosomal storage disorder. Visible here is
the monomer in CPK (Corey, Pauling, Koltun) color.
The 2.1 A resolution X-ray crystal reveals secondary structure consisting of 26 % alpha-helices and 62 % beta-strands. One major and one minor beta-sheet comprise the core of ASA which are largely surrounded by helices. A magnesium atom located at the active site is shown as a reference point throughout the presentation.
Six disulfide bonds exist to stabilize
the ASA monomer. One in the loop connecting the major
and minor beta-sheets, two in an extruded
hairpin and three knotting together the 20
C-terminal amino acids.
Three high mannose type oligosaccharide chains are attached at Asn 158, Asn 184, and Asn 350. The first two N-acetylglucosamine residues attached to Asn 184 are visible. Chains on Asn 158 and Asn 350 are phosphorylated.
In the acidic lysosome, ASA exists as a homo-octamer. Only the monomer
is represented on the left, but important sites of interaction are demonstrated
here. Monomer-monomer interaction is largely
through hydrogen bonding both directly and mediated by water molecules
trapped between monomer surfaces. Dimer-dimer
contact is stabilized by hydrophobic interactions through a long alpha-helix.
Helices of two ASA molecules are arranged
nearly antiparallel to each other and interdigitate via aliphatic
side chains spaced 3 or 4 residues apart along the helix.
pH regulation of dimer-octamer association is thought to be mediated by
protonation/deprotonation of Glu424, which
is shielded in a hydrophobic pocket. At pH
5 Glu424 is protonated and can form intermolecular
H-bond with Phe398O of a second ASA, stabilizing the octamer. At pH 7 Glu424
is deprotonated, the H-bond to Phe398O is sterically unfavorable and the
octamer is destabilized by electrostatic repulsion. In the disassociated
form the Glu424 H-bonds intramolecularly to
Gln460E.