Structural study of the sugar moieties of monoclonal antibodies secreted by human-mouse hybridoma

Six monoclonal antibodies, three each of human IgG1 and IgG2 subclasses, were obtained from human-mouse hybridomas. Structural study of their asparagine-linked sugar chains was performed to elucidate the regulatory mechanism of secreted monoclonal IgG glycosylation. The sugar moieties were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted into radioactive oligosaccharides by NaB3H4 reduction after N-acetylation. Structural study of each oligosaccharide by lectin affinity column chromatography, sequential exoglycosidase digestion, and methylation analysis indicated that almost all of them were biantennary complex-type sugar chains containing Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)GlcNAc as core structures. Bisecting N-acetylglucosamine residue, which is present in human IgG but not in mouse IgG, could not be detected at all. The molar ratio of each oligosaccharide from the six IgG samples was different. However, no subclass specificity was detected except that all IgG1 contained neutral, mono-, and disialylated sugar chains, whereas IgG2 did not contain disialylated ones. The molar ratio of N-acetylneuraminic acid to N-glycolylneuraminic acid was also different for each IgG. All six IgGs contained monoantennary complex-type and high mannose-type oligosaccharides which had never been detected in serum IgGs of various mammals so far investigated. These results indicated that the processing of asparagine-linked sugar chains of IgG is less complete in human-mouse hybridoma than in human or mouse B cells, and that the glycosylation machinery of the mouse cells is dominant in the hybrid cells.     

Comparative study of the asparagine-linked sugar chains of human lipocalin-type prostaglandin D synthase purified from urine and amniotic fluid, and recombinantly expressed in Chinese hamster ovary cells

Lipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaalpha2-->3Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaalpha2-->3Gal and Siaalpha2-->6Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS glycoforms in different tissues.     

Generation of mutated variants of the human form of the MHC class I-related receptor,FcRn, with increased affinity for mouse immunoglobulin G

Much data support the concept that the MHC class I-related receptor FcRn serves to regulate immunoglobulin G (IgG) concentrations in serum and other diverse body sites in both rodents and humans. Previous studies have indicated that the human ortholog of FcRn is endowed with unexpectedly high stringency in binding specificity for IgGs. In contrast to mouse FcRn, which binds promiscuously to IgGs across species, human FcRn does not bind to mouse IgG1 or IgG2a, and interacts weakly with mouse IgG2b. Here, we investigate the molecular basis for this high-level specificity. We have systematically mutated human FcRn residues to the corresponding mouse FcRn residues in the regions that encompass the FcRn-IgG interaction site. Notably, mutation of the poorly conserved residue Leu137 of human FcRn to glutamic acid (L137E) generates a human FcRn mutant that binds to mouse IgG1 and mouse IgG2a with equilibrium dissociation constants of 13.2 microM and 14.4 microM, respectively. From earlier high-resolution structural analyses of the rat FcRn-rat Fc complex, residue 137 of human FcRn is predicted to contact residue 436 of IgG, which can be either His436 (mouse IgG1, mouse IgG2a) or Tyr436 (human IgG1, mouse IgG2b). The simplest interpretation of our data for the L137E mutant is therefore that replacement of the Leu137-Tyr436 (human) by the Glu137-His436 (mouse) pair generates a receptor that can bind to mouse IgG1 and mouse IgG2a. The L137E mutation reduces the affinity of human FcRn for human IgG1 by about twofold, consistent with the introduction of a less favorable Glu137-Tyr436 interaction. However, the analysis of the effects of other mutations on the binding to different IgGs indicates that the contribution to binding of the interaction of FcRn residue 137 with IgG residue 436 can vary. This suggests the existence of distinct docking topologies that are accompanied by variations in contacts between these two residues for different FcRn-IgG pairs. Our observations are of direct relevance to understanding the molecular nature of the human FcRn-IgG interaction. In turn, understanding human FcRn function has significance for the optimization of the serum half-lives of therapeutic and prophylactic antibodies.     

Structural heterogeneity of sugar chains in immunoglobulin G. Conformation of immunoglobulin G molecule and substrate specificities of glycosyltransferases

The heterogeneous asparagine-linked sugar chains of bovine and human immunoglobulins G were separated into 12 components by reversed-phase high performance liquid chromatography, and their structures were determined by 1H NMR spectroscopy. Both immunoglobulin (Ig) G sources contained eight non-bisected biantennary complexes and four bisected biantennary complexes. In the non-bisected sugar chains of bovine IgG, galactosylation of the Man alpha 1-3 branch predominated over that of the Man alpha 1-6, whereas in the bisected complexes galactosylation of the Man alpha 1-6 branch predominated. This difference can be explained by the substrate specificities of the galactosyl-transferases and of the N-acetylglucosaminyltransferase III involved in their synthesis. The sugar chains of human IgG1 differs in the distribution of its galactose residues from bovine IgG and human IgG2. The Man alpha 1-6 branch of all IgG1s was more highly galactosylated than the Man alpha 1-3 branch even in the non-bisected complexes. Such findings are in conflict with the substrate specificities of galactosyltransferases. Whereas these enzymes derivatized more of the Man alpha 1-6 branch of native human IgG1, in denatured protein more of the Man alpha 1-3 branch was galactosylated. Thus, protein conformation may influence the structure of its sugar chains.     

Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity

Antibodies may be viewed as adaptor molecules that provide a link between humoral and cellular defence mechanisms. Thus, when antigen-specific IgG antibodies form antigen/antibody immune complexes the effectively aggregated IgG can activate a wide range of effector systems. Multiple effector mechanisms result from cellular activation mediated through a family of IgG-Fc receptors differentially expressed on leucocytes. It is established that glycosylation of IgG-Fc is essential for recognition and activation of these ligands. IgG antibodies predominate in human serum and most therapeutic antibodies are of the IgG class.The IgG-Fc is a homodimer of N-linked glycopeptide chains comprised of two immunoglobulin domains (Cgamma2, Cgamma3) that dimerise via inter-heavy chain disulphide bridges at the N-terminal region and non-covalent interactions between the C-terminal Cgamma3 domains. The overall shape of the IgG-Fc is similar to that of a "horseshoe" with a majority of the internal space filled by the oligosaccharide chains, only attached through asparagine residues 297.To investigate the influence of individual sugar (monosaccharide) residues of the oligosaccharide on the structure and function of IgG-Fc we have compared the structure of "wild-type" glycosylated IgG1-Fc with that of four glycoforms bearing consecutively truncated oligosaccharides. Removal of terminal N-acetylglucosamine as well as mannose sugar residues resulted in the largest conformational changes in both the oligosaccharide and in the polypeptide loop containing the N-glycosylation site. The observed conformational changes in the Cgamma2 domain affect the interface between IgG-Fc fragments and FcgammaRs. Furthermore, we observed that the removal of sugar residues permits the mutual approach of Cgamma2 domains resulting in the generation of a "closed" conformation; in contrast to the "open" conformation which was observed for the fully galactosylated IgG-Fc, which may be optimal for FcgammaR binding. These data provide a structural rationale for the previously observed modulation of effector activities reported for this series of proteins.     

The structure of unit B-type glycopeptides from porcine thyroglobulin

The structure of Unit B-type glycopeptides (monosialo-type and disialo-type) was investigated by Smith degradation, methyllation, and mass spectral analysis. These glycopeptides contain three peripheral sugar chains. Two are composed of D-galactose residues linked at C-6 and 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and the other is composed of a D-galactose residues linked at C-6, a 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and a D-mannose residue linked at C-2. Most of these peripheral sugar chains are linked to two inner D-mannose residues which are substituted at C-3 and C-6, and constitute branching points. L-Fucose and N-acetyl-neuraminic acid residues are nonreducing terminal groups, and a di-N-acetylchitobiose moiety is linked to an asparagine residue in the peptide moiety. By methylation analysis of the oligosaccharide obtained by hydrazinolysis of the disialoglycopeptide, the L-fucose residues was found to be linked to C-6 of the 2-acetamido-2-deoxy-D-glucose residue linked to the asparagine residue. From these results, and from the previously reported data on the sugar sequence and the anomeric configurations of the linkages between sugar residues, structures for these glycopeptides are proposed.     

Murine alpha-fetoprotein: N-terminal amino acid sequence and C-terminal residue.

We have purified to homogeneity murine alpha-fetoprotein (MAFP) and determined the amino acid sequence of the first twenty-four residues. The N-terminal sequence obtained shows a high degree of homology with human and rat AFP's, but not human or rat albumins. The C-terminal residue is the same as human and “slow” rat AFP, but different from the corresponding albumins. We conclude that the AFP's are derived from homologous genes which are at best distantly related to the ancestral gene for albumin. The single C-terminal residue and N-terminal sequence suggests that the multiple forms of MAFP observed by others are due to carbohydrate micro-heterogeneity.     

Structures of the carbohydrate chains of membrane glycoproteins IIb and IIIa of human platelets

Human platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked carbohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosaccharides released from GPIIb and about 52% of that from GPIIIa, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a beta-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant carbohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

Difference in the sugar chains of two subunits and of isozymic forms of rat kidney gamma-glutamyltranspeptidase

A comparative study by gel-permeation chromatographic analysis of oligosaccharides released from the heavy and the light subunits of rat kidney gamma-glutamyltranspeptidase has revealed that high-mannose-type sugar chains are found only in the heavy subunit, and the nonsialylated and nonfucosylated biantennary complex-type sugar chains are included only in the light subunit. By the same analysis of the oligosaccharide fractions obtained from four isozymic forms of rat kidney gamma-glutamyltranspeptidase, it was found that all these enzymes contain 2 mol of neutral sugar chains but different numbers of acidic sugar chains. The total numbers of sialic acid residues showed a reciprocal relationship to the isoelectric point of each isozymic form, and an increase of 1 mol of sialic acid residue corresponds to a decrease of 0.5 in the value of the isoelectric point.     

Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells

The asparagine-linked sugar chains of natural interferon-beta 1 secreted from human foreskin fibroblasts by poly I:poly C induction and of three recombinant human interferon-beta 1 produced by Chinese hamster ovary cells, mouse epithelial cells (C127), and human lung adenocarcinoma cells (PC8) were released quantitatively as oligosaccharides by hydrazinolysis followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were comparatively analyzed. More than 80% of the sugar chains of natural interferon-beta 1 occur as biantennary complex-type sugar chains, approximately 10% of which contain N-acetyllactosamine repeating structure in their outer chain moieties. The remainders are 2,4- and 2,6-branched triantennary complex-type sugar chains. The sugar chains of the recombinant interferon-beta 1 derived from Chinese hamster ovary cells were very similar to those of its natural counterpart. In contrast, two other recombinant proteins contain quite different sugar chains. The protein derived from C127 cells contains complex-type sugar chains with the Gal alpha 1----3Gal beta 1----4GlcNAc group in their outer chain moieties. Their sialic acid residues occur solely as the Sia alpha 2----6Gal group, where Sia is sialic acid. In contrast, the sialic acid residues of other interferon-beta 1 occur as the Sia alpha 2----3Gal group only. A part of the sugar chains of the protein derived from PC8 cells contains bisecting N-acetylglucosamine residue in addition to the Gal alpha 1----3Gal beta 1----4GlcNAc group.     

Improved Method for Drawing of a Glycan Map,and the First Page of Glycan Atlas,Which Is a Compilation of Glycan Maps for a Whole Organism

Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.     

Structural studies of the carbohydrate moieties of rat kidney gamma-glutamyltranspeptidase. An extremely heterogeneous pattern enriched with nonreducing terminal N-acetylglucosamine residues

The carbohydrate moieties of gamma-glutamyltranspeptidase purified from rat kidney were released as oligosaccharides by hydrazinolysis. Fractionation of the oligosaccharide mixture by paper electrophoresis and Bi-Gel P-4 column chromatography and structural study of each component by sequential exoglycosidase digestion in combination with methylation analysis and periodate oxidation have revealed that it is composed of 23 neutral oligosaccharides, monosialyl derivatives of 67 oligosaccharides, disialyl derivatives of 62 oligosaccharides, and trisialyl derivatives of 5 oligosaccharides. The neutral oligosaccharides are either high mannose type or biantennary complex type, and the acidic oligosaccharides are bi-, tri-, and tetranntennary complex type sugar chains. Most of the complex type sugar chains contain an N-acetylglucosamine residue at the C-4 position of the beta-mannosyl residue of their trimannosyl core. Another characteristic feature of these complex type sugar chains is that they are enriched with nonreducing terminal beta-N-acetylglucosamine residues.     

Structures of the sugar chains of interferon-gamma produced by human myelomonocyte cell line HBL-38

Interferon-gamma produced by the human myelomonocyte cell line HBL-38 contained galactose, mannose, fucose, N-acetylglucosamine, and N-acetylneuraminic acid as sugar components. Sugar chains were liberated from interferon-gamma by hydrazinolysis. Free amino groups of the sugar chains were acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine under new reaction conditions in which no sialic acid residue was hydrolyzed. The pyridylamino (PA-) derivatives of the sugar chains thus obtained were purified by gel filtration and reversed-phase HPLC. Seven major PA-sugar chains were isolated and the structure of each purified PA-sugar chain was identified by stepwise exoglycosidase digestion and 500-mHz 1H-NMR spectroscopy. The results indicated that the structures of the major PA-sugar chains were of the biantennary type, to which 0 to 2 mol of fucose and 1 to 2 mol of N-acetylneuraminic acid were linked as shown below. (formula; see text)     

Fb''2, a new peptic fragment of human immunoglobulin G.

The digestion of a human IgG1 K myeloma protein with pepsin in the presence of 8M-urea was observed to produce a fragment, designated Fb'2, which differed from the products of aqueous peptic digestion and from other characteristic immunoglobulin digestion products. 2. Fragment Fb's was also found when two other IgG1/K proteins were treated similarly. 3. Sedimentation-equilibrium studies showed the mol.wt. of fragment Fb'2 to be 56800. 4. On reduction, two equivalents of each of three peptides were released from fragment Fb's; these were characterized by N- and C-terminal determinations and by amino acid sequencing. 5. Fragment Fb'2 was shown to consist of the constant regions of both light chains, from residue Ile-117 to the C-terminus, and the CH1 domains and hinge region of the heavy chains, from residue Val-113 to residue Met-252, with a gap of five residues within the intrachain disulphide loop, between residues Leu-174 and Tyr-180.     

Structural study of the carbohydrate moieties of two human immunoglobulin subclasses (IgG2 and IgG4)

Asparagine-linked sugar chains were quantitatively released as oligosaccharides from human IgG2 and IgG4 myeloma proteins by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Each oligosaccharide was isolated by serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion and methylation analysis, revealed that all of them were of the bi-antennary complex-type containing Man alpha 1-6(+/- GlcNAc beta 1-4)(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(+/- Fuc alpha 1-6)GlcNAc as core structures, and GlcNAc beta 1-, Gal beta 1-4GlcNAc beta 1- and Sia alpha 2-6Gal beta 1- in their outer chain moieties. However, the molar ratio of each oligosaccharide was different in each IgG sample, indicating that clonal variation is included in the sugar chain moieties of IgG molecules. One of the IgG2 contained four asparagine-linked sugar chains in one molecule, two on the Fc fragment and the remainder on the Fab fragment. The sugar chains in the Fc fragment contained much less galactose as compared with the Fab fragment.     

Comparison of methionine oxidation in thermal stability and chemically stressed samples of a fully human monoclonal antibody

Methionine (Met) oxidation is a major degradation pathway of protein therapeutics. Met oxidation of a fully human recombinant monoclonal antibody was investigated under both chemically stressed conditions using tert-butylhydroperoxide (tBHP) and thermal stability conditions where the sample was incubated in formulation buffer at 25 degrees C for 12 months. This antibody has one Met residue on each of the light chains and four Met residues on each of the heavy chains. In the thermal stability sample, only Met residues 256 and 432 in the Fc region were oxidized to form methionine sulfoxide, while Met residues in the Fab region were relatively stable. The susceptibility of Met residues 256 and 432 was further confirmed by incubating samples with tBHP, which has been shown to induce Met oxidation. Further analysis revealed that the susceptible Met residues of each heavy chain were randomly oxidized in samples incubated with tBHP, while in the thermal stability sample, the susceptible Met residues of one heavy chain were preferentially oxidized.     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.     

Evidence for the existence of O-linked sugar chains consisting of glucose and xylose in bovine thrombospondin.

We have recently discovered unusual sugar chains [xylose-glucose and (xylose)2-glucose] linked to a serine residue in the first epidermal growth factor (EGF)-like domains of human and bovine coagulation factors VII, IX, and protein Z. The sequence surrounding this serine residue has a common -Cys-X-Ser-X-Pro-Cys- structure. Since one (residues 533-538) of the three EGF-like domains found in human thrombospondin contains the conserved sequence, we examined the presence of such O-linked sugar chains in bovine thrombospondin (bTSP) and its 210-kDa fragment. Component sugar analysis after pyridylamination (PA) of the acid hydrolysates of the S-aminoethylated proteins revealed that the proteins contain glucose (Glc) and xylose (Xyl). The oligosaccharide moieties released from intact bTSP by hydrazinolysis followed by pyridylamination were separated into two PA-oligosaccharides by high performance liquid chromatography (HPLC). Component sugar analysis of these PA-oligosaccharides indicated that they consist of Glc and Xyl in molar ratios of 1:1 and 1:2 (or 1:3). The reducing ends of both PA-sugar chains were found to be PA-Glc, as judged from the retention time of the HPLC peak of their hydrolysates. The presence of these PA-sugar chains in bTSP was confirmed by HPLC mapping with two different columns, using standard PA-di- or PA-trisaccharide derived from coagulation factors. From these results, we concluded that bTSP contains O-linked sugar chains consisting of Glc and Xyl in one of its three EGF-like domains.     

Fragments produced by digestion of human immunoglobulin G subclasses with pepsin in urea.

It was previously shown that digestion of human IgG1/kappa myeloma proteins with pepsin in the presence of 8 M-urea produces fragments which differ from other proteolytic fragments of IgG, including those produced by peptic digestion in aqueous buffers. The two large urea/pepsin fragments each consist of three peptides, and together account for all of the constant region of the light chains and most of the constant region of the heavy chains. Myeloma proteins of subclasses IgG2, IgG3 and IgG4 with kappa light chains were digested with pepsin in 8 M-urea, and the resulting fragments compared with those produced from IgG1/kappa proteins. Gel filtration, starch- and polyacrylamide-gel electrophoresis and sequence analysis have shown that the peptides from each subclass are analogous with those from IgG1. A brief investigation of the products of urea/pepsin digestion of myeloma proteins with lambda light chains has shown that in these proteins light-chain cleavage occurs at residue leucine-182, instead of or as well as at residue 117, where cleavage takes place in kappa chains. Comparison of sequences around sites of urea/pepsin cleavage has shown that pepsin has quite restricted specificity under these conditions.