 |
PDBsum entry 1y2m
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Structure-Based chemical modification strategy for enzyme replacement treatment of phenylketonuria.
|
|
|
Authors
|
|
L.Wang,
A.Gamez,
C.N.Sarkissian,
M.Straub,
M.G.Patch,
G.W.Han,
S.Striepeke,
P.Fitzpatrick,
C.R.Scriver,
R.C.Stevens.
|
|
|
Ref.
|
|
Mol Genet Metab, 2005,
86,
134-140.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
Structure-based protein engineering coupled with chemical modifications (e.g.,
pegylation) is a powerful combination to significantly improve the development
of proteins as therapeutic agents. As a test case, phenylalanine ammonia-lyase
(PAL, EC 4.3.1.5) was selected for enzyme replacement therapy in phenylketonuria
[C.R. Scriver, S. Kaufman, Hyperphenylalaninemia:phenylalanine Hydroxylase
Deficiency. The Metabolic and Molecular Bases of Inherited Disease, McGraw-Hill,
New York, 2001, Chapter 77], an inherited metabolic disorder (OMIM 261600)
causing mental retardation due to deficiency of the enzyme l-phenylalanine
hydroxylase (EC 1.14.16.1). Previous in vivo studies of recombinant PAL
demonstrated a lowering of blood l-phenylalanine levels; yet, the metabolic
effect was not sustained due to protein degradation and immunogenicity [C.N.
Sarkissian, Z. Shao, F. Blain, R. Peevers, H. Su, R. Heft, T.M. Chang, C.R.
Scriver, A different approach to treatment of phenylketonuria:phenylalanine
degradation with recombinant phenylalanine ammonia lyase, Proc. Natl. Acad. Sci.
USA 96 (1999) 2339; J.A. Hoskins, G. Jack, H.E. Wade, R.J. Peiris, E.C. Wright,
D.J. Starr, J. Stern, Enzymatic control of phenylalanine intake in
phenylketonuria, Lancet 1 (1980) 392; C.M. Ambrus, S. Anthone, C. Horvath, K.
Kalghatgi, A.S. Lele, G. Eapen, J.L. Ambrus, A.J. Ryan, P. Li, Extracorporeal
enzyme reactors for depletion of phenylalanine in phenylketonuria, Ann. Intern.
Med. 106 (1987) 531]. Here, we report the 1.6A three-dimensional structure of
Rhodosporidium toruloides PAL, structure-based molecular engineering, pegylation
of PAL, as well as in vitro and in vivo PKU mouse model studies on pegylated PAL
formulations. Our results show that pegylation of R. toruloides PAL leads to
promising therapeutic efficacy after subcutaneous injection by enhancing the in
vivo activity, lowering plasma phenylalanine, and leading to reduced
immunogenicity. The three-dimensional structure of PAL provides a basis for
understanding the properties of pegylated forms of PAL and strategies for
structure-based re-engineering of PAL for PKU treatment.
|
|
|
Secondary reference #1
|
|
|
Title
|
|
A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase.
|
|
|
Authors
|
|
C.N.Sarkissian,
Z.Shao,
F.Blain,
R.Peevers,
H.Su,
R.Heft,
T.M.Chang,
C.R.Scriver.
|
|
|
Ref.
|
|
Proc Natl Acad Sci U S A, 1999,
96,
2339-2344.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Figure 1.
Fig. 1. PAL gene from yeast R. toruloides was cloned in
the expression vector pIBX-7 where transcription is controlled
by the strong inducible tac promoter and terminated by the rRNA
transcription terminator sequences rrnBT1 and rrnBT2. LacI^q
represses the tac promoter, and hence isopropyl  -D-thiogalactoside
is required to release it from the promoter. The kanamycin
resistance gene (Kan^R) is included in the construct to allow
selection of cells containing the plasmid.
|
|
Figure 3.
Fig. 3. Injection i.p. of recombinant PAL enzyme reduces
plasma phenylalanine in the ENU2/2 mouse (y axis is logarithmic
scale) over time (x axis) (P < 0.05). Reduction of plasma
phenylalanine by PAL shows a dose-response relationship (z
axis). Data are normalized to the control (sham-treated) values
for each animal at each point. Data depicted are the average of
five paired series. The range of control (100%) values was
390-2,013 µmol/liter for animals receiving 2 units of PAL,
572-1,488 µmol/liter for animals receiving 20 units, and
504-1,474 µmol/liter for animals receiving 100 units.
|
|
|
|
|
|
|
|
|
|
