Glycopeptides from human kappa-chains.
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ABSTRACT:
SUBMITTER: Milstein CP
PROVIDER: S-EPMC1176557 | biostudies-other | 1971 Jan
REPOSITORIES: biostudies-other
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Project description:1. The light chains of human immunoglobulin (Ig) exist in two forms, kappa (type K) and lambda (type L). The two types of chains can be partially separated by taking advantage of the fact that lambda-chains, for the most part, dissociate from reduced Ig at higher pH than do the kappa-chains. The same difference in dissociation of type K and L chains was observed with myeloma IgG and IgA proteins, but not with pathological IgM proteins. 2. When analysed in urea-glycine starch gels, pH7, both kappa- and lambda-chains show ten electrophoretic bands having the same mobilities as those of the whole light-chain subfractions. Normal kappa- and lambda-chains show similar differences in overall amino acid composition to those previously found with myeloma kappa- and lambda-chains and type K and L Bence-Jones proteins.
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Project description:Despite the ubiquitous presence of proteoglycans in mammalian systems, methodologies to synthesize this class of glycopeptides with homogeneous glycans are not well developed. Herein, we report the first synthesis of a glycosaminoglycan family glycopeptide containing two different heparan sulfate chains, namely the extracellular domain of syndecan-3. With the large size and tremendous structural complexity of these molecules, multiple unexpected obstacles were encountered during the synthesis, including high sensitivity to base treatment and the instability of glycopeptides with two glycan chains towards catalytic hydrogenation conditions. A successful strategy was established by constructing the partially deprotected single glycan chain containing glycopeptides first, followed by union of the glycan-bearing fragments and cleavage of the ester-type protecting groups. This work lays the foundation for preparing other members of this important class of molecules.
| S-EPMC4139068 | biostudies-literature
Project description:The arrangement of the disulphide bridges of the major component of the light chains of immunoglobulins (kappa-chains) has been studied in the Bence-Jones proteins. Three disulphide bridges have been found. An interchain bridge at the C-terminus has been shown to occur in the dimers of all the proteins studied and was characterized by symmetrical peptides. In the monomer form, the C-terminal half-cystine of the corresponding peptides was linked to a lone half-cystine residue. A second common disulphide-bridge peptide in which a single amino acid difference could be related to the Inv factors of the individual proteins was found in Bence-Jones proteins and in the kappa-chains of normal and abnormal immunoglobulins. Peptides characteristic of a third disulphide bridge studied in three specimens were found to have differences in some residues, but also striking similarities. A methionine peptide has also been characterized in two specimens as a by-product of the technique employed. It is suggested that a general manner of folding may be a common feature of the heterogeneous population of kappa-chains: one bridge which folds an invariable stretch of the chain, another bridge which folds a stretch that varies from protein to protein, and a bridge at the C-terminus which is the interchain link.
| S-EPMC1270114 | biostudies-other
Project description:IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the ? or ? isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with ? LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with ? LCs. Here, we dissect the domain contributions to Fab stability for both ? and ? LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/C?, and variable domains, VH/V?, within ? LC-containing Fabs is significantly weaker than that of ? LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within ? LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/V? paired with CH1/C? or T cell receptor C?/C?), the major role of the constant domains for both ?- and ?-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/V? and VH/V? scFvs that secreted as a mixture of monomer and aggregates.
| S-EPMC5058631 | biostudies-literature
Project description:Proteoglycans play critical roles in many biological events. Due to their structural complexities, strategies towards synthesis of this class of glycopeptides bearing well-defined glycan chains are urgently needed. In this work, we give the full account of the synthesis of syndecan-3 glycopeptide (53-62) containing two different heparan sulfate chains. For assembly of glycans, a convergent 3+2+3 approach was developed producing two different octasaccharide amino acid cassettes, which were utilized towards syndecan-3 glycopeptides. The glycopeptides presented many obstacles for post-glycosylation manipulation, peptide elongation, and deprotection. Following screening of multiple synthetic sequences, a successful strategy was finally established by constructing partially deprotected single glycan chain containing glycopeptides first, followed by coupling of the glycan-bearing fragments and cleavage of the acyl protecting groups.
| S-EPMC5110403 | biostudies-literature
Project description:The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAβ3Galβ3Galβ4Xylβ-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcβ4GlcAβ3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.
| S-EPMC4762621 | biostudies-literature
Project description:BackgroundKappa free light chains (KFLC) are a promising new biomarker to detect neuroinflammation. Still, the impact of pre-analytical effects on KFLC concentrations was not investigated.MethodsKFLC concentrations were measured in serum and cerebrospinal fluid (CSF) of patients with a newly diagnosed multiple sclerosis (MS) or clinically isolated syndrome (CIS) before (n = 42) or after therapy with high-dose methylprednisolone (n = 65). In prospective experiments, KFLC concentrations were analyzed in the same patients in serum before and after treatment with high-dose methylprednisolone (n = 16), plasma exchange (n = 12), immunoadsorption (n = 10), or intravenous immunoglobulins (n = 10). In addition, the influence of storage time, sample method, and contamination of CSF with blood were investigated.ResultsPatients diagnosed with MS/CIS and treated with methylprednisolone showed significantly lower KFLC concentrations in serum as untreated patients. Repeated longitudinal investigations revealed that serum KFLC concentrations continuously decreased after each application of methylprednisolone. In contrast, other immune therapies and further pre-analytical conditions did not influence KFLC concentrations.ConclusionOur results show prominent effects of steroids on KFLC concentrations. In contrast, various other pre-analytical conditions did not influence KFLC concentrations, indicating the stability of this biomarker.
| S-EPMC7226742 | biostudies-literature
Project description:BackgroundThe determination of kappa free light chains (KFLC) in cerebrospinal fluid (CSF) is an upcoming biomarker for the detection of an intrathecal immunoglobulin synthesis. Since renal function impairment leads to altered serum KFLC and albumin concentrations, interpretation of KFLC in CSF may be influenced by these parameters.MethodsIn this two-center study, the influence of renal function (according to the CKD-EPI creatinine equation) on KFLC and albumin concentrations was investigated in patients with "physiological" (n = 139), "non-inflammatory" (n = 146), and "inflammatory" (n = 172) CSF profiles in respect to the KFLC index and the evaluation in quotient diagrams in reference to the hyperbolic reference range (KFLC IF).ResultsAll sample groups displayed declining KFLC indices and KFLC IF values with decreasing renal function (P-values between <.0001 and .0209). In "inflammatory" CSF profile samples, 15% of the patients presented a KFLC index <5.9 while 10% showed an intrathecal KFLC fraction below QKappa(lim), suggesting possible false negative KFLC results.ConclusionsThe influence of renal function should be considered while interpreting KFLC results in patients with neuroinflammatory diseases. The interpretation of KFLC in quotient diagrams is less susceptible to renal function impairment than the KFLC index and should be preferentially used.
| S-EPMC8619759 | biostudies-literature
Project description:Free light chains (FLC) are a promising biomarker to detect intrathecal inflammation in patients with inflammatory central nervous system (CNS) diseases, including multiple sclerosis (MS). The diagnostic use of this biomarker, in particular the kappa isoform of FLC ("KFLC"), has been investigated for more than 40 years. Based on an extensive literature review, we found that an agreement on the correct method for evaluating KFLC concentrations has not yet been reached. KFLC indices with varying cut-off values and blood-CSF-barrier (QAlbumin) related non-linear formulas for KFLC interpretation have been investigated in several studies. All approaches revealed high diagnostic sensitivity and specificity compared with the oligoclonal bands, which are considered the gold standard for the detection of intrathecally synthesized immunoglobulins. Measurement of KFLC is fully automated, rater-independent, and has been shown to be stable against most pre-analytic influencing factors. In conclusion, the determination of KFLC represents a promising diagnostic approach to show intrathecal inflammation in neuroinflammatory diseases. Multicenter studies are needed to show the diagnostic sensitivity and specificity of KFLC in MS by using the latest McDonald criteria and appropriate, as well as standardized, cut-off values for KFLC concentrations, preferably considering non-linear formulas such as Reiber's diagram.
| S-EPMC8622045 | biostudies-literature
Project description:Three type K Bence-Jones proteins have been fully reduced and carboxymethylated with high-specific-activity iodo[(14)C]acetate. A tryptic digest and a chymotryptic digest of each protein were fractionated on a Sephadex column and the radioactive peptides were purified by paper electrophoresis. All the proteins studied had five unique carboxymethylated cysteine sequences. Three of these were identical with the exception of a single substitution, and two had some variations. The common peptides could be placed in the C-terminal half of the molecule. The variations around the other two half-cysteine residues provided information about the nature of the variability of the primary sequence of immunoglobulin kappa-chains. The results are consistent with the hypothesis that the chains derive from a common ancestor by somatic mutation of a small number of genes or by gene doubling and selection in the course of evolution. The isolation of the N-terminal peptide in methionine-containing Bence-Jones proteins is also described.
| S-EPMC1270115 | biostudies-other