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Articles |
1
Biochimie A, Hôpital Bichat-Claude Bernard AP-HP, 75877 Paris Cedex 18, France.
2
Laboratoire de Biochimie Générale, UFR
Sciences Pharmaceutiques et Biologiques–Paris XI, 92260
Châtenay-Malabry Cedex, France.
3
Faculté de Pharmacie, Université Paris V,
75270 Paris Cedex 06, France.
a Address correspondence to this author at: Laboratoire de Biochimie A, Hôpital Bichat-Claude Bernard, 46, Rue H. Huchard, 75877 Paris Cedex, France. Fax 33-1-4025-8821; e-mail nathalie.seta{at}bch.ap-hop-paris.fr
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Abstract |
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 Top Abstract IntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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Approach: This review summarizes those diseases that are known to be the result of an inherited or acquired glycoprotein oligosaccharide structural alteration and that are diagnosed in blood or urine by chemical characterization of that oligosaccharide alteration.
Content: The biochemical synthesis steps and catabolic pathways important in determining glycoprotein function are outlined with emphasis on alterations that lead to modified function. Clinical and biochemical aspects of the diagnosis are described for inherited diseases such as I-cell disease, congenital disorders of glycosylation, leukocyte adhesion deficiency type II, hereditary erythroblastic multinuclearity with a positive acidified serum test, and Wiskott-Aldrich syndrome. We also review the laboratory use of measurements of glycoforms related to acquired diseases such as alcoholism and cancer.
Conclusions: Identification of glycoprotein glycoforms is becoming an increasingly important laboratory contribution to the diagnosis and management of human diseases as more diseases are found to result from glycan structural alterations.
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Introduction |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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This review addresses the inherited and acquired diseases in which biological diagnosis is based on alterations in the oligosaccharide structure of glycoproteins secreted into blood and urine and confirmed by measuring the activity of cellular enzymes involved in the altered glycosylation pathway (1).
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O- and N-Oligosaccharide Structures of Glycoproteins |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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In O-glycosylated proteins, the oligosaccharides range in size from 1
to >20 sugars, displaying considerable structural (and antigenic)
diversity. Moreover, these oligosaccharides are not uniformly
distributed along the peptide chain; they are clustered in heavily
glycosylated domains. N-Acetylgalactosamine (GalNAc) is
invariably linked to Ser or Thr (Fig. 1
). Mannose residues are never detected in mature O-glycans.
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N-Oligosaccharides have a common core structure of five sugars and
differ in their outer branches. The first sugar residue,
N-acetylglucosamine (GlcNAc) is bound to Asn included in a specific
tripeptide sequence (Asn-X-Thr or Ser). N-Oligosaccharides are
classified into three main categories: high mannose, complex, and
hybrid (Fig. 1
). High-mannose oligosaccharides have two to six
additional mannoses linked to the pentasaccharide core and forming the
branches. Complex-type oligosaccharides have two or more branches, each
containing at least one GlcNAc, one Gal, and eventually one
sialic acid (SA); they can be bi-, tri-, or tetraantennary (Fig. 1
).
Hybrid oligosaccharides contain one branch that has the complex
structure and one or more high-mannose branches. Glc residues are never
detected in mature complex N-oligosaccharides. Serum glycoproteins
mostly consist of complex type N-oligosaccharides. O- and
N-oligosaccharide chains may occur on the same peptide core.
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Biosynthesis Steps of O- and N-Oligosaccharides |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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). The glycan chain then grows by the addition of GlcNAc, Gal,
and Fuc residues in the medial Golgi. Sialylation continues throughout
the trans Golgi. There are several possible pathways to construct
O-glycans, depending on the substrate specificity and intracellular
arrangement of glycosyltransferases. However, it is far less complex
than the processing of N-oligosaccharides
(4)(5).
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The biosynthesis of N-oligosaccharides (Fig. 3
) begins in the ER with a large precursor oligosaccharide that
contains 14 sugar residues. The inner five residues constitute the
core, which is conserved in the structure of all N-linked
oligosaccharides. This precursor is linked to dolichol pyrophosphate,
which acts as a carrier for the oligosaccharide. This lipid-linked
oligosaccharide is transferred "en bloc" to an Asn residue on the
growing polypeptide chain (6). While the nascent
glycoprotein is still in the rough ER, all three Glc residues and one
mannose residue are removed by specific glycosidases, producing
an oligosaccharide with 10 residues instead of 14. The maturation of
the N-oligosaccharides takes place in the Golgi complex. This pathway
involves a coordinated and sequential set of enzymatic reactions, which
remove and add specific sugar residues. The enzymes involved
(glycosidases and glycosyltransferases) are located in the cis, medial,
and trans Golgi (7). The reaction product of one enzyme is
the substrate for the next. When present, SA residues are always at the
terminal nonreducing ends of oligosaccharides. The high-mannose and
hybrid oligosaccharides appear as intermediates along the processing
pathway. The complex type is the mature form of N-linked
oligosaccharides.
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Catabolic Pathways of N- and O-Oligosaccharides |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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illustrates the degradation pathway of N-oligosaccharides,
which begins with the elimination of Asn by
N-aspartyl-ß-glucosaminidase and is achieved by a
ß-mannosidase. In contrast to N-oligosaccharides, the catabolism of
O-oligosaccharides is poorly documented and likely involves sequential
and ordered events catalyzed by specific osidases, which remain to be
characterized (8)(9).
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Catabolism of oligosaccharides also occurs in the cytosol. This concerns mostly the catabolism of oligomannosides, which originate either from the ER or from dolichol intermediates. There is intense intracellular trafficking of free mono- and oligosaccharides among the ER, cytosol, and lysosomes, which involves specific membrane carriers.
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Microheterogeneity of Glycans |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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These glycoforms can be classified into restricted subsets of glycoproteins, using tools such as lectins (12), which recognize specifically a sugar residue or a small oligosaccharide structure. Affinity chromatography and crossed immuno-affinoelectrophoresis currently are used for biological diagnosis (13).
Using lectins greatly helps biologists to identify glycoforms and to detect variations in the relative proportions of glycoforms for a specific glycoprotein. For example, concanavalin A (Con A), the best-known plant lectin, specifically recognizes the trimannosidic structure of a N-glycoprotein if mannose residues are accessible (14). Schematically, Con A interacts with biantennary oligosaccharides but not with tri- and tetraantennary structures (15).
Fig. 5
illustrates how crossed immuno-affinoelectrophoresis in the
presence of Con A, a simple and rapid method, can provide information
about glycosylation. On the basis of the four Con A patterns of
1-acid glycoprotein (AGP) shown in Fig. 5
(from a healthy adult, a pregnant woman, the umbilical cord blood from
a healthy newborn, and from an infected newborn suffering from
listeriosis), it appears that (a) hormonal changes during
pregnancy alter glycosylation of AGP with an increase of unretained
forms of AGP; (b) AGP is not transferred from the mother to
the fetus through the placental barrier; and (c) in response
to infection, the fetus is able to produce some mediators that alter
its AGP patterns without altering the AGP pattern of its mother.
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Functions of Oligosaccharides in Glycoproteins |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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Few general rules that relate oligosaccharide structure to function have been established. Tridimensional conformation allows oligosaccharides to be recognized by receptors and to mediate events such as cell trafficking, cell adhesion, and biological half-life. These biological events involve biochemical interactions between oligosaccharides as ligands and proteins named lectins (18). Ashwell and Morell (19), when discovering the hepatic asialoglycoprotein receptor, identified the first mammalian lectin. It recognizes the ultimate Gal residue of desialylated serum glycoproteins and rapidly clears them from blood by endocytosis.
Cell and cell interactions involving a specific oligosaccharide structure are also well documented in the literature. The well-known recruitment of neutrophils to sites of inflammation as part of the cell’s defense against bacterial infection is initiated by the interaction of a specific oligosaccharide present at the surface of a cell with its lectin counterpart, which is called a selectin (20).
Numerous carbohydrate-binding specificities involved in infection and toxicity have been identified for a wide variety of microorganisms (21).
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Inherited Diseases |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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i-cell disease, or leroy disease
I-Cell disease is a severe and rare genetic disease, inherited in
an autonomic recessive manner and caused by the lack of Golgi GlcNAc
phosphotransferase (9)(22)(23)(24)(25).
Clinical features.
Typical patients present with mental
retardation, coarse facial features with gingival hyperplasia, and
short-trunk dwarfism; they generally die in the first decade of life
(23).
Biochemical aspects.
Whereas the oligosaccharides on secretory
and membrane N-glycoproteins are processed to complex-type units,
mannose residues on lysosomal enzymes become phosphorylated. The
phosphorylation of mannose residues is a two-step procedure involving
two separate enzymes in the cis Golgi: GlcNAc phosphotransferase and
GlcNAc1 phosphodiester N-acetylglucosaminidase. GlcNAc
phosphotransferase recognizes only lysosomal enzymes as substrates and
catalyzes the addition of a GlcNAc phosphate residue to mannoses of the
branches. N-Acetylglucosaminidase removes the GlcNAc group,
leaving the phosphate attached to the carbon of the mannose (Fig. 6
). Subsequently, the phosphorylated lysosomal enzymes bind
tightly and specifically to a receptor, the mannose 6-phosphate
receptor, a transmembrane protein of the trans Golgi, which directs the
enzymes into vesicles coated with clathrin. After depolymerization, the
uncoated vesicles fuse with late endosomes in which the low pH allows
the phosphorylated enzymes to dissociate from their receptors. Finally,
transport vesicles containing the lysosomal enzymes fuse with
lysosomes.
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The lack of the first enzyme, GlcNAc phosphotransferase, causes I-cell disease, a lysosomal storage disease, which leads to a deficiency of multiple enzymes in fibroblasts and macrophages. Lysosomal enzymes with normal enzymatic activity are no longer directed to the lysosomes, but are secreted into the extracellular space. Alternative mechanisms must operate in other cell types to target lysosomal enzymes into lysosomes. Thus, hepatocytes, Kupffer cells, and leukocytes from patients with I-cell disease contain nearly normal concentrations of enzymes despite their deficiency in phosphotransferase activity.
Laboratory diagnosis.
Biochemical diagnosis is based on
strongly increased plasma lysosomal enzyme activities and is confirmed
by the decrease of GlcNAc phosphotransferase activity in fibroblasts.
The gene encoding GlcNAc phosphotransferase has not been cloned. No
defects in GlcNAc1 phosphodiester N-acetylglucosaminidase or
in the mannose 6-phosphate receptor have been reported.
congenital disorders of glycosylation
Another newly delineated group of carbohydrate-related genetic
diseases is termed congenital disorders of glycosylation (CDG;
previously known as carbohydrate-deficient glycoprotein syndrome)
(26). Two types are described, based on the enzymatic
defect: type I corresponds to the CDG that involve enzymatic steps up
to the assembling of the glycoprotein; type II are the CDG in which
processing is involved. The most common one is CDG Ia (>300 patients
worldwide), which is attributable to a deficiency in cytosolic
phosphomannomutase activity in the metabolism of mannose
(27).
Clinical features.
CDG other than type Ib are multisystemic
disorders involving neurological dysfunction. CDG Ia infants have a
typical morphology with abnormal distribution of subcutaneous fat and
inverted nipples.
Clinical symptoms of CDG Ib are limited to intestinal and hepatic disease.
Biochemical aspects.
Phosphomannomutase catalyzes the
transformation of mannose 6-phosphate to mannose 1-phosphate, which is
then transformed to GDP mannose, a precursor of mannose for the
biosynthesis of N-glycoproteins (Fig. 7
). Phosphomannomutase deficiency (CDG Ia) leads to the synthesis
of glycoproteins present in the serum as a mixture of various
glycoforms: normally glycosylated, and partially or totally devoid of
oligosaccharide chains. This is illustrated with transferrin (TRF) in
Fig. 8
. The gene encoding for the incriminated phosphomannomutase-2 is
located on chromosome 16p13 (28), and numerous mutations and
one single-pair deletion have been identified recently (29).
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CDG Ib is caused by defective phosphomannose isomerase activity, which reversibly converts fructose 6-phosphate into mannose 6-phosphate. Mannose treatment has been used successfully to correct clinical and biochemical symptoms (30).
The very recently described CDG Ic (or V) is caused by a defect in dolichyl-P-Glc:Man9GlcNAc2-PP dolichyl glucosyltransferase activity, which adds the first of three glucosyl residues to the nascent N-glycoproteins in the ER (31).
Serum glycoprotein glycosylation alterations observed in CDG Ib and Ic are similar to those in CDG Ia.
CDG type II (two described patients) is related to a deficiency in GlcNAc transferase II (cis Golgi), which leads to the synthesis of complex N-glycoproteins devoid of one branch (32)(33).
Laboratory diagnosis.
CDG are diagnosed by
demonstrating the presence of abnormally glycosylated serum
glycoproteins. Several electrophoretic methods may be used:
(a) isoelectrofocusing, which detects the different
glycoforms as a function of their SA content [Serum TRF, which was for
the first time studied by Stibler et al. (34) in relation to
alcoholism, is currently analyzed (35).]; and
(b) Western blotting, which detects the different glycoforms
as a function of their molecular weights. Fig. 9
shows typical Western blot profiles of serum TRF, haptoglobin,
AGP, and 1-proteinase inhibitor from a healthy
adult and a CDG Ia patient (36).
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The enzymatic deficiencies and the corresponding gene mutations can be demonstrated in leukocytes and in cultured skin fibroblasts from patients (27)(29)(30).
CDG type II also is easily diagnosed by Western blot analysis of serum TRF and by demonstrating the GlcNAc transferase II deficiency and its related gene mutations (33)(36).
leukocyte-adhesion deficiency type ii
Leukocyte-adhesion deficiency type II (LAD II) is a rare severe
immunological disease (37).
Clinical features (38)(39).
LAD II is clinically similar to the well-described LAD I, in that is
also involves severe mental retardation, short stature, and dysmorphic
features. The two syndromes differ in the molecular basis of their
adhesion defects. LAD I is caused by a deficiency in the CD18
integrin-adhesion molecule, whereas LAD II neutrophils are devoid of
sialyl Lewis x, which is a ligand for the selectin family.
Biochemical aspects.
The selectin family, including L-, E-,
and P-selectins (40), mediates rolling and tethering of
leukocytes along the walls of the microvasculature before adhesion and
extravasation. The selectins contain lectin-like domains that bind
fucosylated oligosaccharides such as sialyl Lewis x
[SA(2,3),Galß1,4(Fuc (1,3))GlcNAc].
In addition to being sialyl Lewis x negative, LAD type II patients
express oligosaccharide structures that are devoid of Fuc(1,2)Gal,
Fuc(1,4)GlcNAc, and Fuc(1,6)GlcNAc. These fucose residues are the
products of at least four different fucosyltransferases, but it is
unlikely that this genetic defect is a fucosyltransferase deficiency.
The reported impaired fucosylation could be attributable to a defect
either in the transport of fucose to the Golgi lumen or in any enzymes
in the synthetic pathway of GDP-Fuc, which is the substrate for the
fucosyltransferases. It appears to be caused by a defect in the
conversion of GDP-Man to GDP-Fuc. Thus, the lack of sialyl Lewis x on
the neutrophils would be secondary to a general fucose deficiency.
Laboratory diagnosis.
Biochemical diagnosis could be
established by evaluating the absence of the sialyl Lewis x epitope on
neutrophil membranes using specific anti-sialyl Lewis x antibodies.
hereditary erythroblastic multinuclearity with a positive acidified
serum test
Hereditary erythroblastic multinuclearity with a positive
acidified serum test (HEMPAS) (41), also called congenital
dyserythropoietic anemia type II
(42)(43), is a rare autosomal recessive disorder
caused by membrane abnormality, with >300 known patients, and is a
multifactorial disease.
Clinical features.
Congenital dyserythropoietic anemia type II
patients suffer from a long-life anemia, hepatosplenomegaly, liver
hemosiderosis, and cirrhosis.
Biochemical aspects.
HEMPAS erythrocyte band 3 shows a
complete absence of poly N-acetyllactosamine. The disease
might be heterogeneous because the primary defect is not totally clear.
Indeed, some cases are related to GlcNAc transferase II deficiency
(44). Other patients had been identified with normal GlcNAc
II activity but no detectable activity in -mannosidase II. Both
enzymes are necessary to convert complex immature N-glycans to complex
mature N-glycans. In addition, the defect seems to be limited to
hematopoietic cells, which does not totally correspond to the clinical
features.
Studies on linkage analysis and allele segregation showed that there
was no linkage between congenital dyserythropoietic anemia type II
phenotype and the chromosomal regions containing the candidate genes
that code for GlcNAc transferase II and -mannosidase II. Ialoscin et
al. (45) and Gasparini et al. (46) suggest that
the disease is most likely attributable to a defect of a
transcriptional factor regulating both enzymes.
Laboratory diagnosis.
Biochemical diagnosis can be made by
showing the presence of abnormally glycosylated band 3 in the
electrophoretic pattern of erythrocyte membranes of patients, using
sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
wiskott-aldrich syndrome
Wiskott-Aldrich syndrome (WAS) is an X-linked, rare disease
characterized by eczema, thrombocytopenia, and immunodeficiency
(1)(47).
Clinical features.
The clinical features begin early in life
and include recurrent infections, bleeding, and eczema. The prognosis
is grave. Circulating platelets are decreased in number and size. IgM
concentrations usually are below normal, IgG concentrations are normal,
and IgA and IgE concentrations are high. Monoclonal gammopathy is
common.
Biochemical aspects.
WAS is caused by defects of the
WAS gene (48), which encodes the WAS
protein, involved in proliferation and differentiation of the
hematopoietic progenitors (49). A specific defect in
O-linked glycans in lymphocytes has been reported with the abnormal
expression on WAS peripheral blood mononuclear cells of a highly
O-glycosylated integral membrane, sialoglycoprotein, CD43. N-Linked
glycans are normal. In addition, spectacular differences in the
activities of two O-glycosyltransferases, core 2 GlcNAc transferase and
-2,6-sialyltransferase, are related to the abnormal O-glycan
synthesis (50)(51).
Other than the deficiency of galactosyltransferase I involved in the assembly of glycosaminoglycans (52), WAS is the only genetic disease involving abnormal O-glycosylation identified at present.
Laboratory diagnosis.
Biochemical diagnosis can be established
by evaluating the O-glycosylation of CD43 in blood mononuclear cell
membranes.
glycoproteinoses
The common feature of these diseases is the genetic deficiency of
any one of the lysosomal glycosidases involved in the
catabolism pathway of glycoproteins (single-entity incidence between 1
in 100 000 and 1 in 250 000) (53).
Clinical features.
Most of these diseases are characterized by
different clinical phenotypes, depending the age of the patient. The
clinical feature in the infantile form is most often dominated by
neurologic symptoms. Retinal abnormalities are frequent.
Biochemical aspects.
The deficiency of one of the lysosomal
enzymes involved in the catabolic pathway of glycoproteins leads to the
accumulation of undegraded oligosaccharides in lysosomes and increased
urinary excretion of carbohydrate material produced by the cytolysis of
sick cells. An accumulation of glycolipids and mucopolysaccharides is
also observed because the oligosaccharide moiety is degraded by the
same set of enzymes.
Laboratory diagnosis.
The biochemical diagnosis is based on
chromatography of urinary oligosaccharides. Low cell-specific
glycosidase activity confirms the results obtained by chromatography.
Mutations or deletions of some of the genes that code specific enzymes
have been identified and can be demonstrated in some glycoproteinoses.
Table 1
illustrates the main biochemical features of these diseases.
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Acquired Diseases |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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carbohydrate-deficient transferrin and chronic alcohol drinking
Biochemical aspects.
Human TRF is a serum glycoprotein that
exists in many different forms determined by genetic polymorphisms,
iron saturation, and oligosaccharide composition. TRF contains two
N-linked complex-type major biantennary oligosaccharides
(54). In normal plasma, the major glycoform is
tetrasialylated (80%) with a small proportion of pentasialylated and
trisialylated forms (15%). The hexa-, di-, mono-, and asialylated
subtypes are present in very low concentrations.
In chronic alcoholics, the plasma concentration of tetrasialylated TRF decreases, whereas the concentration of disialylated TRF increases (55). When alcoholism is associated with malnutrition and/or hepatocellular deficiency, the percentages of mono- and asialylated TRF also increase.
Laboratory diagnosis.
Serum carbohydrate-deficient
transferrin, which represents partially and totally
desialylated TRF, currently is measured to detect and monitor chronic
drinkers.
Isoelectric focusing was first used to separate serum TRF isoforms, but the current method uses anion-exchange chromatography to separate serum TRF isoforms, which are then measured by RIA or enzyme immunoassay (56)(57). Commercial kits (CDTect EIA) are available.
alkaline phosphatase in bone and hepatobiliary diseases
Biochemical aspects.
Human alkaline phosphatase (ALP) exists
as several isoforms that may appear in plasma under different
conditions of health and disease (58). Four
structural genes that encode ALP have been sequenced. The
tissue-nonspecific ALP gene is expressed in osteoblasts,
hepatocytes, kidney, early placenta, and other cells. The ALP
isoforms from bone and hepatocytes, which are coded by the
same gene, are differently glycosylated, depending the
glycosyltransferases in both cell types. Healthy adults generally have
approximately equal plasma activities of the liver and bone glycoforms.
The source of an increase in serum ALP can be documented by identifying
the ALP oligosaccharide moiety.
Laboratory diagnosis.
Wheat germ agglutinin is a lectin that
interacts preferentially with bone ALP. Its use in association with
affinity techniques leads to an increase in the specificity and
sensitivity of total serum ALP measurement
(59)(60). Clearly, serum bone ALP is a very
reliable marker of bone formation. However, this method was used first
some years ago and is being replaced by immunological assays that use a
monoclonal antibody that recognizes the bone glycoform.
malignant diseases
Tumor-associated antigens often involve carbohydrates and are
termed "TACAs" for tumor-associated carbohydrate antigens, which
are found in both glycolipids and N- and O-linked oligosaccharides of
glycoproteins (61)(62)(63). These tumor-associated carbohydrate
antigens seem to function mainly as adhesion molecules based
on either carbohydrate interactions or carbohydrate selection
interactions. Some of these antigens are found exclusively in
mucin-type glycoproteins and are known as T, sialyl T, Tn, and sialyl
Tn, each defined by a specific monoclonal antibody (Fig. 10
). Their specific or high degrees of expression in certain
types of cancer prompted researchers to evaluate their potential use as
diagnostic and/or prognostic tools. It appeared that if their
"diagnostic value" for cancer detection was low, they could have
"prognostic value", particularly in the early stages of tumor
development. The clinical usefulness of their expression in tumors or
in sera of cancer patients is under investigation in comparison studies
with available tumor markers.
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Oligosaccharide alterations are not restricted to surface antigens, but
are also detected in serum glycoproteins. -Fetoprotein (AFP), an
oncofetal glycoprotein, in particular has been studied. Altered and
different glycosylation of serum AFP has been found in serum from
patients suffering from hepatocellular carcinoma and seminomatous
germ-cell tumors (64). Specific lectins, which recognize
these altered oligosaccharides, are used in affinity electrophoresis,
which is easy to perform routinely.
Biochemical aspects.
AFP, one of the major plasma
glycoproteins in early embryonic life, is synthesized in the yolk
sac and then by the liver (64). In an advanced stage of
pregnancy, the concentration of liver-synthesized AFP in fetal serum
decreases, whereas the albumin concentration increases. After birth,
the serum AFP concentration becomes very low. In hepatocellular
carcinomas and in germ-cell tumors, AFP is re-expressed, and its serum
concentration greatly increases.
During pregnancy, AFP is also subjected to variations in the oligosaccharide moiety, giving different glycoforms, the proportions of which depend on the cell types secreting AFP. In adults, glycoforms of re-expressed AFP specific for hepatic regeneration (65) and yolk sac-derived tumors (66) are identified by the presence of Fuc on the first GlcNac of the core or an additional bisected GlcNAc on the first Man, respectively.
Laboratory diagnosis.
The use of a combination of two lectins,
Con A and Lens culinaris agglutinin (LCA) allows
laboratorians to distinguish between benign liver disorders and
hepatocellular carcinoma and between hepatic and germ-cell tumors. The
presence of Fuc on the first GlcNAc residue of a biantennary
oligosaccharide largely favors the interaction of LCA with such
oligosaccharide structures, whereas the presence of a bisected GlcNAc
residue at the trimannosidic core dramatically decreases the
interaction of biantennary oligosaccharides with Con A. Briefly, serum
AFP from hepatocellular carcinoma reacts with LCA and Con A, whereas
AFP from yolk sac tumors binds LCA and does not react with Con A (Fig. 11
). Several studies investigating diagnostic tools for
hepatocellular carcinoma have shown that the percentage of LCA-reactive
serum AFP is more sensitive than the serum AFP concentration itself.
This is particularly interesting in the monitoring of patients with
chronic liver diseases (67) because the incidence of
hepatocellular carcinoma in association with cirrhosis is high.
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The simultaneous determination of Con A- and LCA-reactive glycoforms in crossed immuno-affinoelectrophoresis for AFP is also particularly useful for differentiating seminomatous from nonseminomatous germ-cell tumors in neonates and infants in whom the serum AFP concentration is in the upper physiological range (68).
other diseases
Changes in the oligosaccharide moieties of glycoproteins are
linked to many other disorders. The immune complexes from patients with
rheumatoid arthritis are particularly rich in asialo-agalactosyl IgG,
and these changes in IgG glycosylation are a consistent feature of
patients with rheumatoid arthritis (69).
The oligosaccharide profile of acute phase proteins is modified in
inflammatory disorders (70)(71), without any
relation to the serum concentrations of these glycoproteins during
acute inflammation. In several serum glycoproteins, such as AGP, TRF,
and 2-HS glycoprotein, during acute
inflammation the number of branches is reduced, whereas during chronic
inflammation, the number increases. These changes are easily
demonstrated by crossed immuno-affinoelectrophoresis in the presence of
Con A.
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Conclusion |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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Glycobiology is a very active field of research that may lead to new approaches in the diagnosis and prognosis of human diseases.
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Footnotes |
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1-acid glycoprotein; CDG, congenital disorders of glycosylation; TRF, transferrin; LAD, leukocyte adhesion deficiency; HEMPAS, hereditary erythroblastic multinuclearity with a positive acidified serum test; WAS, Wiskott-Aldrich syndrome; ALP, alkaline phosphatase; AFP, -fetoprotein; and LCA, Lens culinaris agglutinin. 
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References |
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TopAbstractIntroductionO- and N-Oligosaccharide...Biosynthesis Steps of O-...Catabolic Pathways of N-...Microheterogeneity of GlycansFunctions of Oligosaccharides in...Inherited DiseasesAcquired DiseasesConclusionReferences |
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1-acid glycoprotein. III. Polymorphism of 1-acid glycoprotein and the partial resolution and characterization of its variants. Biochemistry 1967;1:959-966.
-glutamyl transferase and mean corpuscular volume. Arch Intern Med 1995;155:1907-1911.
[Abstract]
-Fetoprotein: 25 years of study. Tumor Biol 1989;10:63-74.
-fetoprotein purified from human ascites fluid. Cancer Res 1980;40:4276-4281.
-fetoprotein purified from a human yolk sac tumor and its reactivity with concanavalin A. Tumor Biol 1984;5:33-40
, GalNAc; 
, GlcNAc; 
, galactose; •, mannose; 
, fucose 
, SA; ~, polypeptide chain.
, SA; ~, polypeptide chain. Dol, dolichol;
P, phosphate.
