In Vitro Expression analysis: an update and brief commentary on the
Paula J. Waters (IVE curator) - June 17th 2002.
Data concerning in vitro expression analysis of
naturally-occurring mutations in the human PAH gene have recently been
extensively updated online, to include a large number of findings published over
the last year. As of June 2002, the searchable table entitled "in
vitro expression (human)" summarises analysis of 81 different PAH
mutations, the vast majority of which are missense alleles. There are currently
a total of 225 individual records of analysis, reflecting the fact that
frequently a given mutation may have been studied in multiple expression systems
and/or by different research groups.
for expression analysis
In vitro expression analysis has been driven by three linked
- To confirm that a "disease-associated" mutation is truly pathogenic
(rather than a coincident benign change).
- To assess the severity of a mutation's impact (and aid the
correlation of PAH genotype with hyperphenylalaninemia phenotype).
- To help understand how a mutation exerts its deleterious effects
(elucidate the molecular mechanisms involved).
While all three continue to be valuable lines of investigation, over recent
years the emphasis has shifted heavily towards the third of these goals. In the
process, our understanding of the mechanisms has reshaped our thinking on how
best to identify, and attempt to quantitate, a mutation's pathogenicity.
Various expression systems have been applied to study PAH mutations,
by placing the mutant (and wild-type) cDNAs into plasmid vectors and introducing
these into host cells. The systems include:
- Transiently transfected human or other mammalian host cells - being
probably the closest available approximation to the in vivo
- Transformed bacterial (E. coli) cells - where high protein
production often permits enzyme purification and thus its detailed
- in-vitro transcription-translation systems - allowing
synthesis of radiolabelled PAH, the fate of which can be followed
- Two-hybrid systems (in yeast or mammalian cells) - allowing the
study of interactions between PAH monomers.
Often the study of a single mutation in multiple complementary expression
systems allows the piecing together of a coherent picture of the mutation's mode
of action and the severity of its impact on the function of the expressed
(It must however be noted that these classic IVE expression systems
only examine the effects of nucleotide changes in the context of cDNA, not
of genomic DNA. Therefore any effects of apparent missense or silent
substitutions upon RNA splicing in vivo will not be observed using these
included in the IVE(human) table in PAHdb
Each record in the IVE (human) table has twelve listed parameters, as
- Mut. name: The mutation name, using the trivial (amino acid)
- IVEhID: A unique identifier for the individual record of expression
- Plasmid: The plasmid vector used.
- Host: The host cell type used.
- PAH Enzyme: Refers to PAH enzyme activity measured in
cell lysates (as a percentage of the wild-type value).
- PAH Immuno: Refers to the amount of (immunoreactive) PAH protein
present in cell lysates (again, as a percentage of the wild-type
value). Frequently a decrease in cellular PAH enzyme activity
is in fact accounted for simply by a decrease in the PAH protein
- PAH mRNA: Refers to PAH mRNA levels in cell lysates
(as a percentage of the wildtype value).In practice this has not yet been
found to be decreased for any mutation studied in these systems, thus is not
the explanation for observed decreases in PAH protein levels. Note:
Columns 5,6 and 7 (Pah Enzyme, PAH Immuno, PAH mRNA) were inadvertently
omitted from the print versions of the IVE(human) tables included in recent
Newsletters; they are however present and correct in the data viewable
- Spec. act.: Refers to enzyme specific activity, i.e. the ratio of PAH
activity / PAH protein. Where this is less than 100% relative to
wild-type, it is highlighted; this finding is especially pertinent in
mammalian cell systems, since it may provide a pointer to mutations which
affect PAH enzyme activity independently of an effect on cellular PAH
- Note: Refers to methodological details which have a bearing on
interpretation of the observed results.
- Addit. Observ.: Includes additional observations on the effects of
a mutation, including insights into mechanisms.
- Date entered: The date of entry online.
- Reference: The full citation for the original research; usually a
published paper, occasionally the record of a direct submission to the PAH
As the sophistication and complexity of IVE data has tended to
increase, the conversion of literature data to a simple format compatible with
their concise summary in tabular form has become progressively more challenging!
The interested visitor to the PAHdb IVE tables is therefore always
referred back to the primary citation for further details.
messages from IVE (human) data
Accumulated data from various researchers together illustrates some important
- A common type of mechanism now appears to be implicated in the
pathogenicity of numerous PAH missense mutations. Such mutations
promote misfolding of the PAH monomer and oppose correct assembly of
monomers into the native tetrameric enzyme. The resulting structural
aberrations trigger cellular defences, provoking accelerated degradation of
the abnormal protein by proteases. The intracellular steady-state levels of
the mutant PAH protein are therefore reduced, leading to an overall
decrease in phenylalanine hydroxylation within cells, and thus to
- The effects of such mutations may well be modulated in vivo by
modification of the cellular handling (folding, assembly and degradation) of
the mutant enzyme. This has major implications, first for our understanding
of genotype-phenotype correlations (and their imperfections), secondly for
possibilities of novel therapeutic approaches.
- Certain PAH mutations have more direct adverse effects on enzyme
activity - for instance, by affecting binding of substrate or cofactor to
the enzyme's catalytic or regulatory sites, or by affecting residues with
other critical roles in the catalytic process.
- The pathogenicity of some PAH missense mutations appears to reflect
a combination of different mechanisms acting in concert. Dissecting out
their relative in vivo significance is an ongoing challenge.
mutations and rat PAH mutations
PAHdb also contains two smaller tables of data related to in vitro
expression analysis. (These are pre-queried, rather than searchable by
individual mutation). The "in vitro expression (artificial)"
table documents artificially-created mutations in the human PAH gene (not
corresponding to naturally-occurring mutations). The "in vitro
expression (rat)" table documents artificially-created mutations in
the rat PAH gene (a few of which do correspond to natural mutations in
the homologous human gene). For both of these datasets, the rationale for the
studies is slightly different than for studies on naturally-occurring mutations
- where the emphasis is on understanding the direct relevance of those mutations
to the associated disease phenotypes observed in patients. The artificial
mutations (both human and rat) instead have been designed specifically to
examine structure / function relationships in the wild-type enzyme, by targeting
residues hypothesised to have key functional roles. Analysis accordingly has
focussed on the properties of the isolated mutant protein (usually purified from
expression in E. coli), rather than on the fate of the protein in its
"in vivo" cellular context. Design of these two IVE
tables therefore differs somewhat from that of the primary IVE (human)
The IVE (artificial) and IVE (rat) tables should also be
updated online by the end of July 2002.