Table Of ContentLIST OF COMMITTEE MEMBERS
N. AXELSEN J. HOBBS
Statens Seruminstitut Westminster Medical School
Treponematoses Department University of London
COPENHAGEN, DENMARK LONDON, GREAT BRITAIN
R. BALLIEUX T. HUISMAN
Academisch Ziekenhuis Medical College of Georgia
Immunologisch Laboratorium Sickle Cell Center
UTRECHT AUGUSTA (GEORGIA)
THE NETHERLANDS U.S.A.
B. BOWMAN E. LÜSCHER
Dept. of Cellular & Structural Theodor Kocher Institute
Biology University of Bern
University of Texas BERN
SAN ANTONIO (TEXAS) SWITZERLAND
U.S.A.
P. MASSON
P. BURTIN Université de Louvain
Institut de Recherches Dept. de Méd. Expérimentale
Scientifiques sur le Cancer BRUSSELS, BELGIUM
VILLEJUIF, FRANCE
V. OREKHOVICH
A. CARBONARA Inst. of Medical Chemistry
Istituto di Genetica Medica Academy of Medical Sciences
dell'Università di Torino MOSCOW
TORINO, ITALY U.S.S.R.
H. CLE VE H. PEETERS
Institut für Anthropologie Director
und Human Genetik Institute for Medical Biology
MUNICH BRUSSELS, BELGIUM
WEST GERMANY
D. POULIK
Z. DISCHE William Beaumont Hospital
College of Physicians and ROYAL OAK (MICHIGAN)
Surgeons of Columbia University U.S.A.
NEW YORK, U.S.A.
F. PUTNAM
P. GRABAR Div. of Biological Sciences
Institut Pasteur Indiana University
PARIS, FRANCE BLOOMINGTON (INDIANA)
U.S.A.
H. HARBOE
Harboes Laboratorium P. G. RIGHETTI
COPENHAGEN, DENMARK Universita di Milano
MILANO, ITALY
H. HIRAI
Tumour Laboratory A. SCHADE
TOKYO, JAPAN 3901 Indian School Road
ALBUQUERQUE
S. HJERTEN
(NEW MEXICO)
Institute of Biochemistry
U.S.A.
University of Uppsala
UPPSALA, SWEDEN
PROTIDES OF THE
BIOLOGICAL FLUIDS
PROCEEDINGS OF THE THIRTY-THIRD
COLLOQUIUM, 1985
Edited by
H. PEETERS
Director
Institute for Medical Biology
Brussels, Belgium
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PREFACE
The Arne Tiselius Memorial Lecture was held by Prof. F. Putnam under the topic Progress
in Plasma Proteins. This was his second A.T.M.L.. The title of the previous one being
Alpha, Beta, Gamma, Omega? The Past, Present and Future of Plasma Proteins. The difference
in flavour between the two titles indicates the importance of the new developments.
As usual three topics were selected : one in the biochemical, one in the clinical and one
in the technical field.
The biochemical topic was concerned with Plasma Protein Genes. The location of genes on
the chromosome, the pathology and variance of several genes and lastly gene expression
were considered in the papers presented. It is obvious that protein chemistry is shifting
from the protein to the responsible gene and this will exert a profound influence on our
approach of protein anomalies.
The proteins of the nucleus and more in particular the detection of antibodies to nuclear
constituents (Antinuclear Antibodies or ANA) was the clinical topic. A wealth of new infor
mation proved the liveliness of this field of investigation which is soon to reach the
detection of specific antibodies to well defined nuclear antigens. Detection methods and
assays were extensively described.
The tradition of the Colloquia requests a chapter on Technical advances and sure a rich
harvest of papers in this domain was the source of fruitful discussions.
Three days of intensive work have thus covered three fields of protein chemistry and again
opened new avenues for future research. These proceedings are an excellent postgraduate
teaching book on protein science.
A CKNOWLEDGEMENTS
We, the members of the Scientific Committee of the XXXIIIrd Colloquium, wish to thank the
Burgomaster and the Aldermen of the City of Brussels for the reception at the Town Hall.
Prof. A. Bollen, , Prof. N. Harboe, Prof. S. Hjertén, Dr. R. Humbel, Prof. F. Putnam, Dr. F.
Ramlau, Prof. P. Righetti and Dr. K. Schäfer Nielsen have been responsible for the concept
and establishment of the different parts of the program.
We thank Miss G. Peeters and Miss M. Declerck for their help in the practical organisation
of the meeting.
The proceedings are being published with a subsidy of the Belgian Ministry of Education.
xxin
PROGRESS IN PLASMA PROTEINS:
STRUCTURE, FUNCTION, AND GENES
FRANK W. PUTNAM
Department of Biology, Indiana University, Bloomington, IN 47405, USA
I. INTRODUCTION: NEW DIRECTIONS IN PLASMA PROTEIN RESEARCH
Much of my early research was influenced by the work of Arne Tiselius, so I was
privileged to give the Second Arne Tiselius Memorial Lecture at the 21st Colloquium in 1973.
I am doubly honored to give the 14th Tiselius Lecture at this, the 33rd Colloquium. I entitled
my Second Tiselius Lecture "Alpha, Beta, Gamma, Omega: The Past, Present, and Future of
Plasma Proteins" (Putnam, 1974). In it I reviewed the history of electrophoresis, I surveyed
the status of knowledge of the plasma proteins, and I predicted the direction of future
progress. The direction was right, but who then could correctly predict the explosive
development of new knowledge and methods? Progress in plasma proteins is ever accelerating
and threatens to overwhelm us by its very volume; yet, simultaneously it excites us to press
forward for new discoveries. It will be no easy task for me to fulfill my title "Progress in
PI asma Proteins." To simpli fy it, I will emphasi ze my own interest in structural studies;
furthermore, for background and literature references I wi11 refer you to my recently
published Volume IV of The Plasma Proteins, which I use as the basis for the review part of
my lecture (Putnam, 1984ä]7
In the past decade great progress has been made in knowledge of the plasma proteins and
of their role in health and disease. New directions have been undertaken that have led to
disuse or abandonment of classical methods for characterization of the plasma proteins,
including methods developed by Tiselius and Svedberg such as free electrophoresis and
analytical ultracentrifugation, which gave rise to the now somewhat vestigial nomenclature of
many of the protein components of plasma. The advances include better methods for
identification, characterization, and isolation of the individual proteins, and ultrasensitive
and precise techniques for their quantitation. Progress in knowledge of the structure of
plasma proteins is rapidly advancing due to enhanced interest and to the development of
powerful new methods, such as HPLC, protein microsequencing, and gene cloning and sequencing.
More than 100 plasma proteins have been characterized. Although immunoglobul in sequences
dominate the databases, the primary structure is now known for at least 40 other plasma
proteins, and the carbohydrate structure for 25. The genes for about 40 have been cloned and
the cDNA sequence and genomic structure reported in many cases, and also the chromosomal
location of the genes. At the same time that such rapid progress was being made in
determining the structure of the polypeptide components of proteins, new developments
facilitated research on the structure and function of the ol igosaccharides. The number of
plasma glycoproteins whose oligosaccharides have been completely characterized has increased
from about 5 to some 25 (Baenziger, 1984). Concurrently, much new knowledge has developed
about the mechanism of protein glycosylation and secretion, and interest has quickened in the
interaction of oligosaccharides with cell receptors.
II. PRIMARY STRUCTURE OF THE PLASMA PROTEINS
Amino Acid Sequence Analysis. After automatic techniques for determination of protein and
peptide sequences became widely available, research on the primary structure of plasma
proteins accelerated. The increase in amino acid sequence data for immunogl obul ins of all
species attained an exponential slope by 1970 and continued unabated with the advent of rapid
3
4 FOURTEENTH ARNE TISELIUS MEMORIAL LECTURE
nucleotide sequencing around 1980 (see Fig. 3, Putnam, 1984b). Increasingly, the primary
structures of plasma proteins are being inferred from the results of gene cloning and
nucleotide sequencing especially for those that have important biological activities, but that
are present in trace amounts and are hard to purify. However, it is notable that the amino
acid sequences of the light and heavy chains of all five classes of human immunoglobulins were
first determined by methods of protein chemistry, culminating with the entire covalent
structure of IgD in 1982 (Takahashi _et aj_., 1982; Putnam et al., 1982). Indeed, as of early
1985, only partial gene sequences had been reported for human IgA, IgM, and IgD.
Although the exponential trend in sequence data for immunoglobulins continues unabated,
the past decade has witnessed a remarkable increase in the number of other plasma proteins for
which the entire amino acid sequence has been determined. This is charted in Fig. 1. By
early 1985, not only had the primary structures been determined for all the known classes and
subclasses of human and mouse immunoglobulins and for some Ig classes of several other
species, but also for about 50 other plasma proteins, largely human and bovine. The roster of
complete structures includes the majority of the most abundant plasma proteins; others are
under intensive study. The structures of very few plasma proteins that can be purified and
have known function have eluded the protein chemist, and many of the missing ones are being
pursued by the molecular biologist. Prominent among those still missing are some of the
complement components and some of the blood coagulation proteins, but these are under
intensive study by both the protein and the gene approach, as will be reported at this
Colloquium.
Fig. 1. Increase in amino acid
sequence data (excluding immuno-
globulins) for all plasma pro
teins of all species (updated
from Fig. 4, p. 22 of Putnam,
1984b). The dates for the com
pletion of the sequence of rep
resentative human plasma pro
teins are indicated by standard
symbols for the proteins. For
reference purposes, the Greek
letters at the abscissa identify
the dates when the complete
amino acid (protein) sequences
were reported for the human
immunoglobulin light (κ, λ) and
heavy (γ, μ, ε, αΐ9 α2, δ)
chains. The curve initiating
in 1980 is for amino acid
sequences inferred from nucleo
tide sequences. An asterisk
denotes proteins for which the
gene sequence was reported prior
to the protein sequence. Note
that prior to 1983 a-fetopro-
tein (AFP) was the only such
case, but in 1984 the data
deduced from gene sequences
increased wery rapidly.
Gene Cloning and Nucleotide Sequences. Although the primary structures of most of the
proteins identified in Fig. 1 were first determined by methods of protein sequencing, there
PROGRESS IN PLASMA PROTEINS 5
has recently been a great increase of data inferred from the nucleotide sequences. This
complementary new approach first showed its power in the discovery of the introns and exons of
the interrupted genes of immunoglobulins. Now, nucleotide sequencing is being widely applied
to genes for other plasma proteins. This has led to knowledge of pre- and propeptide
sequences, to correlation of introns with domain structure, and to knowledge about the loci
and organization of the genes. In principle, the amino acid sequence can be deduced for any
protein for which the gene can be identified and cloned; however, protein sequences, partial
or complete, are often needed to help in the design of a probe for a gene or to identify a
gene product.
As the program of this Col loquium shows, there has been a great influx of molecular
biologists into the study of plasma proteins. The reason is that plasma proteins offer
several practical advantages for gene cloning and sequencing. Other attractions are
anthropomorphic curiosity of Man in the study of Man, and the clinical significance and actual
or potential therapeutic value of human plasma proteins. These latter factors add commercial
incentives that have attracted the new biotechnology companies, which have become major
contributors to the field since 1983. There are several practical advantages: 1) Except for
immunoglobulins, the majority of plasma proteins are synthesized specifically and often in
high amount in 1 i ver, and human 1 i ver DNA 1 i bra ri es are readi lyavailable. 2). For many
human plasma proteins the complete or partial protein sequences are already known. 3)
Antisera are available commercially for about 70 human plasma proteins. 4) Finally, a great
deal is already known about the genetics of human plasma proteins, and kindreds are available
for many genetic variants or hereditary deficiencies. The coincidence of these factors has
resulted in a second-stage exponential increase in information on gene cloning, nucleotide
sequencing, genomic organization, and chromosomal gene location of the plasma proteins.
The flood of new structures that have resulted from protein sequencing is being qreatly
accelerated and even surpassed by nucleotide sequencing (see Fig.l). This tide would
overwhelm the protein chemist were it not for the databases that developed concurrently, e.g.,
Barker et al., 1984; Kabat et_ aj_., 1983. In the research conducted in my laboratory, we have
found that the powerful computer programs of the NBRF-PIR database (formerly "Atlas of Protein
Sequence and Structure") permit rapid retrieval of data and identification of homology among
sequences from regularly updated databases. Later I wi 11 mention a surprising discovery of a
new family of plasma proteins comprising ceruloplasmin and blood coagulation factors VIII and
V. Without the computer methods for sequence retrieval and comparison the totally unexpected
relationship of these very large proteins would not have been discovered. As important and
exciting as these advances are, we should not lose sight of the fact that acquisition of
structural data is only taxonomy if not accompanied by correlation with biological function
and genetics.
III. THREE-DIMENSIONAL STRUCTURE AND ULTRASTRUCTURE OF PLASMA PROTEINS
Three-Dimensional Structure. The ever-increasing success of determining the primary
structure of plasma proteins by protein sequencing or of deducing it from nucleotide sequences
stands in sharp contrast with the slow pace of determining the three-dimensional structure of
plasma proteins by X-ray crystallography, which is the only means of doing it. One reason for
the slow progress is that plasma proteins are large (about 60 kDa or greater), and the
difficulty of crystal 1ographic analysis increases disproportionately with size. Secondly, as
discussed below, many plasma proteins are polymeric and some have bizarre ultrastructures.
Finally, most plasma proteins are glycoproteins, and some have up to 30% carbohydrate; this
adversely affects crystal 1 ographic analysis. In fact, human IgG is one of the very few
glycoproteins from any source, for which the three-dimensional structure has been reported,
but IgG has only one carbohydrate per γ chain, and the structure had to be determined on the
Fc fragment.
Excluding Fab and Fc fragments and light chains of immunoglobulins, about 20 human plasma
proteins have been crystallized. Yet, until recently prealbumin was the only intact plasma
protein among about 200 proteins from many sources for which atomic coordinate data at high
resolution had been reported. However, in 1984 Loebermann et al. reported the crystal
0
structure at 3 A resolution of human a·,-anti trypsin ( a^-proteinase inhibitor) which has
three carbohydrate groups and an M = 54,000.
r
In contrast to the meager results for other plasma proteins, much has been published
about the three-dimensional structures of immunoglobulins (Amzel and Poljak, 1979; Davies and
Metzger, 1983). Yet, all the data are for human myeloma proteins of the IgG class or its Fab
or Fc fragments, or κ or λ light chains (Bence Jones proteins) (see Table III, Putnam, 1984b).
No high-resolution structures have yet been reported for the other immunoglobul in classes
6 FOURTEENTH ARNE TISELIUS MEMORIAL LECTURE
(IgM, IgA, IgD, and IgE), and only low-resolution data have yet appeared for monoclonal
antibodies. Nonetheless, X-ray crystal 1ographic analysis of IgG and its fragments has
essentially defined the main topographical features of the structure of antibodies and of the
combining site in rather fine detail. Because of the diversity of antibody structure and the
nearly infinite number of possible combining sites, a thousand more structures could still be
determined. Each might add more to the fabric of our knowledge, but it is unlikely that any
would greatly change current concepts of the architecture of antibodies.
Ultrastructure. Because of the large size of some plasma proteins, electron microscopy
has recently proven useful in a few instances for visualization of the shape and domain
structure despite the low electron density of the proteins (Table IV of Putnam, 1984b).
However, earlier studies of IgG have not been followed up for IgA, IgD, and IgE. Electron
microscopy has been most useful in the study of the pentameric structures of C-reactive
protein (CRP) and of the plasma amyloid P-component both of which have a pentagonal structure.
Electron microscopy is also being used to study the assembly of the complement complexes and
has revealed a remarkable structure of the Clq component. The ultrastructure of the C4b
binding protein is just as bizarre and is described graphically as having "an unusual spider
like structure" composed of thin, flexible units called "tentacles," each of which binds one
C4b fragment. The ninth and last component of complement (C9) polymerizes as a dodecahedron
forming a doughnut-shaped tubular structure resembling the membrane attack complex of
complement. These observations should remind us that many soluble proteins in plasma only can
exert their physiological functions in mul timolecular complex assemblies that often are
transient and may effect their action either at a cell surface or in solution.
IV. HEREDITARY DEFICIENCIES AND GENETIC POLYMORPHISM OF HUMAN PLASMA PROTEINS
Hereditary Deficiencies of Human Plasma Proteins. Because of increasing emphasis on the
genetic diseases of man, many hereditary deficiencies of human plasma proteins have been
discovered and are being carefully investigated. Some of these are listed elsewhere (Table V,
Putnam 1984b). The number of blood coagulation disorders identified with hereditary
deficiencies of specific proteins has expanded greatly from the several classical hemophilias
to at least a dozen well-defined disorders, some of which will be described at this
Colloquium. There is now much excitement and considerable optimism about commercial attempts
to clone the human genes for blood coagulation proteins, particularly for Factor VIII (AHF,
antihemophi1ic factor). Successful cloning could provide a source of AHF for component
replacement therapy that would avoid transmission of infectious hepatitis or acquired
immunodeficiency syndrome (AIDS). This would also open up the possibility of gene insertion
in patients with hemophilia A (the most common coagulation disorder).
Genetic Polymorphism of Human Plasma Proteins. The search for genetic variants of human
plasma proteins continues both because of interest in the possibility of finding abnormal
forms and because of the value of plasma protein polymorphisms as markers for studies of
population genetics. Unlike the situation with abnormal hemoglobins, few abnormal forms of
human plasma proteins have been identified and then implicated as associated with or causally
related to pathological conditions. However, an inherited polymorphism resulting in multiple
alleine forms is now recognized in at least 50% of the major plasma proteins (Table VI,
Putnam, 1984b). More than 20 genetic variants differing in electrophoretic mobility have been
reported for each of three proteins: serum albumin, a ^-antitrypsin, and transferrin.
Ceruloplasmin, haptoglobin, and a ,-acid glycoprotein each exhibit about ten variant forms.
Allelic forms have been identified either electrophoretical ly or seroloyical ly for a number of
other plasma proteins. There are numerous allotypic and isotypic forms of human
immunoglobul ins (Putnam, 1977; Fudenberg et_ £]_., 1984).
Although there is evidence for many variants of plasma proteins, the structural change
has been identified in only a few cases even though the complete amino acid sequence and
sometimes also the gene sequence have been reported for the proteins involved. The main
reason for this failure is the large size of most pi asma protein polypeptide chains, which
precludes identification of a single variant peptide by traditional peptide-mapping
techniques. However, recently, we have developed a procedure for making a peptide profile of
large proteins such as cerul opl asmin by use of HPLC (Takahashi _et_ aj_., 1985c). The HPLC
profile method may permit identification and isolation of variant peptides in genetic forms of
ceruloplasmin, or in cerul oplasmin from patients with Wilson's disease. Later in this
Colloquium we describe application of this method to study of heavy-chain disease proteins and
to genetic variants of human serum albumin. A different approach to be reported at this
Colloquium by Huchenq and others is the use of restriction enzyme fragment length polymorphism
to identify transferrin variants.
PROGRESS IN PLASMA PROTEINS 7
The structural change has been identified in some genetic variants of plasma proteins.
Yet, although the total number of genetic variants of human plasma proteins described to date
probably exceeds several hundred, a link to a functional disorder has been established in only
a few cases. Coagulation disorders offer the best examples. Nonfunctional forms of blood-
clotting proteins are suspected in several diseases such as the various hemophilias, but in
most cases the nonfunctional protein and the putative structural change have not been
identified. However, the abnormal fibrinogens described in this Colloquium by Henschen offer
examples of proteins where structural variation has been associated with dysfunction. Another
consequence of a mutation in structure may be a greater tendency for aggregation or increased
susceptibility to enzymatic cleavage. Either one or both of these phenomena may be the cause
of a form of familial systemic amyloidosis, in which a single amino acid substitution in
prealbumin is believed to be involved in the mechanism of amyloid fibril formation (Dwulet and
Benson, 1984). Genetic studies of a prealbumin variant will be presented to us by Sarai va et_
al. Failures in postsynthetic processing of genetic variants have been identified in several
cases such as Proalbumin Christchurch which retains the N-terminal propeptide. A similar
failure in processing appears to be the defect in Tangier disease. Recent findings on the
structural abnormalities in apolipoproteins illustrate some of the powerful new approaches to
the study of inherited defects in lipid metabolism. Fortunately, at least three speakers will
present their work on human apolipoprotein genes to us.
V. CHROMOSOMAL MAPPING, GENOMIC ORGANIZATION, AND SITE-DIRECTED MUTAGENESIS OF HUMAN PLASMA
PROTEINS
Chromosomal Mapping. An area that is advancing rapidly, and which will be introduced by
Bowman in this Colloquium is chromosomal mapping and elucidation of the gene structure of the
plasma proteins. A major goal of modern genetics is the complete chromosomal mapping of the
entire human genome, accompanied by the cloning of genes of interest. Already, the locus of
many enzymes has been identified, particularly for some associated with hereditary deficiency
diseases. Recently, genes for human clotting factors and plasminogen tissue activator have
been cloned with therapeutic and economic goals in view. These and other examples will be
described at this Colloquium by several groups of workers.
In the past 3 years, much effort has been directed toward unraveling the complex al leiic
structure of the HLA system of the human major histocompatibi1ity complex (MHC). In fact,
human MHC genes, which are on chromosome 6, have already been mapped, cloned, and sequenced.
As Campbel 1 et_ άλ_. wi 1 1 report at thi s Col 1 oqui um, both the MHC of the mouse and the MHC of
man contain loci for certain complement components such as C2, C4, and factor B. In man,
chromosome 6 also is the locus of blood coagulation factor XIII. Altogether, genes for more
than 25 human plasma proteins have already been mapped to a particular chromosome. About half
of these are components of either the complement system or the immunogl obulin family. Most of
the results have been obtained in the past 2 years, and the pace of research is increasing
rapidly, as today's session on plasma protein genes will indicate.
Genomi c Organization. The immunogl obul in gene system, which has been particularly well
studied both in man and the mouse, has been reviewed in depth by several authors such as Honjo
(1983) and will be described here by Celia Mil stein. The system contains three separate gene
loci; in the human genome the κ, λ, and H (heavy) chain genes are on chromosomes 2, 22, and
14, respecti vely. Studies of the mol ecular biology of the immunoglobuIi n gene system are
helping greatly to elucidate the factors controlling the normal rate of biosynthesis of the
five classes of immunoglobuli ns and the pathological changes that occur in lymphomatous
diseases.
To date, no comparable understanding of the gene organization and the factors controlling
gene expression has been achieved for other plasma protein systems such as complement, blood
coagulation, and 1ipoproteins. However, one interesting development is the discovery of the
proximity and similarity of the genes for serum albumin and a -fetoprotein (AFP). As
described in this Colloquium by Dugaiczyk, the two proteins belong to the same gene family and
exhibit a close relationship in both their protein structure and in the arrangement of the
coding segments (exons) of the DNA of their genes. Furthermore, in the mouse and presumably
also in man the albumin and AFP genes are in tandem only 13.5 kilobases apart and are
transcribed from the same strand of DNA. This may explain their somewhat reciprocal prenatal
and postnatal expression. Another example is the mapping of the transferrin gene to human
chromosome 3. As Yang will report to us, this is the same chromosome to which the transferrin
receptor has been assigned and also the melanoma antigen p97, which is homologous to
transferrin (Yang ^t_ al., 1984).
Gene cloning also demonstrates that some proteins that have a two-chain structure after
