Commentary
In the
era of bioinformatics, big data and high throughput techniques it is quite
tempting to forget the advantage of the good old techniques such as affinity
chromatography, in the search of yet unknown protein molecules. Not so long ago
this very efficient and straightforward technique yielded in quite a short time
numerous bioactive molecules thus opening new fields of basic research. And I
specifically refer to ligand affinity chromatography. This type of separation
is based on unique interaction between the target analyte and a ligand coupled
covalently to a resin [1,2]. It is a simple,
rapid, selective, and efficient purification procedure of proteins. Tens of
thousands fold purification is achieved in one step, which is crucial when an
extremely rich sources of naturally occurring proteins such as human urine or plasma
or spinal fluid are used [3].
The
biological activity of the isolated proteins is retained thus function is
revealed concomitantly with the isolation. Prior to the completion of the
genome project this method facilitated rapid and reliable cloning of the
corresponding gene. Upon completion of this project, a partial protein sequence
is enough for retrieving its complete mRNA and hence its complete protein
sequence. This method is indispensable for the isolation of both expected (e.g.
soluble receptors) but mainly unexpected, unpredicted and very much surprising
binding proteins found by serendipity. Practically, no other approach would
yield the latter [4]. In the group of expected such
proteins are the two receptors for TNF [5,6]
originally named TNF binding proteins (TBPI and TBPII), the Type I interferon
receptor [7], the Type II interferon receptor
and the IL-6 receptor [8]. The unexpected group
of proteins include the unique IL-18 Binding Protein (IL-18BP) [9], the IL-32 binding protein- Proteinase 3 [10] and the heparanase binding protein, the resistin [11].
In the
case of TBPI, the ligand affinity chromatography was a one-step purification
procedure, compared to three inefficient purification steps used beforehand to
purify the TBPI (CM-Sepharose, cation exchange HPLC, anion exchange HPLC) [5]. Moreover, this affinity column made all the
difference since it yielded two TNF binding proteins, the TBPI and the TBPII.
The TBPII [6] would have never been discovered
by the multistep chromatographic procedure used to purify the TBPI, since its
chemistry is different from that of TBPI and it is not retained by the first
purification column used for TBPI, the CM-sepharose. Type I interferon was
discovered in 1957. Note that it took 37 years to isolate its receptor and not
before ligand affinity chromatography was employed [7],
though many laboratories all over the world made attempts to isolate it all
these years. The discovery of the soluble Type I interferon receptor enabled the
identification of its cell-surface counterpart, the ligand binding chain of
Type I interferon receptor, later on named IFNAR2.
In our
hands, a search for the IL-18 receptor failed but the binding protein that was
found instead was a big reward. Encoded by a separate gene, IL-18BP [9] deviates from the classical definition of soluble receptors,
namely, it does not correspond to the extracellular ligand-binding domain of
the IL-18 receptor but rather could be added as a member of a separate family
of rare secreted proteins that bind the same ligand as canonical soluble
receptors do, but are encoded by a different gene (e.g. osteoprotegerin, [12]). The exceptionally high affinity of this IL-18BP
decoy protein to its target protein, the IL-18 [13],
promised its success as a drug that needs no modifications to prolong its half-
life time.
This
characteristic had already been revealed during the process of its
purification, since it smeared though harsh elution conditions were used to
take it off the ligand affinity column instead of coming off as a sharp peak. Yet
additional quite surprising proteins discovered using ligand affinity chromatographies
include the enzyme Proteinase 3 (PR3) and resistin. PR3 was isolated upon a
search for IL-32 receptor and named IL-32 binding protein [10]. PR3, a neutrophil granule serine protease, is
the major antigen for autoantibodies in the systemic vasculitic disease,
Wegener's granulomatosis. This disease can be fatal and its treatment requires
long-term immunosuppressant. The linkage to IL-32, found by serendipity, may
one day shed light on the mechanism of action of this disease.
The
pro-inflammatory cytokine resistin was isolated in an attempt to isolate the
heparanase receptor and was named heparanase binding protein [11]. Resistin, initially described as a rodent
adipokine, is predominantly macrophage-derived in humans and thought to link
between inflammation, metabolic diseases and possibly tumorigenesis. Heparanase,
among others, is strongly implicated in cancer metastasis and is up-regulated
in essentially all human tumors examined. Though resistin and heparanase may
have some tangential biological functions, the mechanism of the observed
unexpected association between those two remains obscure.
Years
later a number of these proteins translated into drugs. It was the TBPII, the second
soluble receptor for TNF, namely the one which was isolated by serendipity that
became a blockbuster drug Etanercept (EnbrelTM).
It is since then prescribed to millions of patients to treat rheumatoid
arthritis, psoriasis and other chronic inflammatory conditions. The use of IL-18BP
in particular conditions is an example for personalized medicine. In 2015, on a compassionate basis, IL-‐18BP (Tadekinig
alfa®) saved a life of a baby born
with a mutation in the inflammasome [14]. This
NLRC4 mutation causes an over expression of IL-18 and results in what is known
as Macrophage Activating Syndrome (MAS). MAS is a life-threatening sepsis-like
condition and is also a complication of cancer, autoimmune diseases and
inflammation. IL-18BP is currently in phase III clinical studies for MAS, Hemophagocytic
Lymphohistiocytosis (HLH) and the systemic autoinflammatoryStill’s disease.
The concept of “Proteome”
was introduced more than 10 years ago, followed by large-scale studies of
protein expression, localization, activities, and interactions, leading to
extensive research and technology development. Proteomics is expansively
applied in many areas, ranging from basic research, various disease and tumor
diagnoses and biomarker discovery, to therapeutic applications. Several
proteomics approaches have been developed for protein separation and
identification and for the characterization of protein function and structure. Affinity
chromatography and mass spectrometry are among the indispensable methods that
enabled this meteoric progress.
short commentary