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Paul A Ramsland

Publications and source records attributed to Paul A Ramsland.

At least 19 recordsLinked to original sources
PubMed2026

Comparative metagenomic analysis of microbial communities: unravelling microbial communities from the great Rann of Kachchh and coastal saltpans, Gujarat, India.

Hypersaline environments exhibit extreme physiochemical conditions yet support diverse microbial communities. These communities are not only ecologically important but also possess substantial potential for biotechnological exploitation. In this study, we employed a comparative metagenomic approach to assess microbial diversity using two distinct methodologies: (1) direct DNA extraction from raw sediment, and (2) DNA extraction following halophilic enrichment in selective media. Sediment samples were collected from multiple sites and pooled together within the Rann of Kachchh and close-by saltpans and were analysed using 16S rRNA sequencing coupled with bioinformatics pipelines. The results revealed pronounced differences in microbial community composition between the two approaches. Raw sediment samples exhibited significantly higher alpha diversity, with dominant taxa including Halobacterota, Cyanobacteria, and Desulfobacterota, with a substantial proportion of unclassified genera. In contrast, enriched samples were dominated by fast-growing, culturable genera such as Halobacterium, Alkalibacillus, and Candidatus haloredivivus. Principal Coordinate Analysis (PCoA) of beta diversity demonstrated distinct clustering between raw and enriched communities, even within samples from the same sites, underscoring the selective bias introduced by enrichment procedures. These findings emphasise that the methodological choice strongly influences the observed microbial diversity. The aim of this study was to compare microbial community composition in raw hypersaline sediments and enrichment cultures using metagenomic sequencing, to evaluate how enrichment selectively favours specific halophilic taxa. This comparative approach allows identification of the microbial groups that rapidly proliferate under controlled hypersaline conditions, thereby complementing direct environmental sequencing. By integrating both direct and enrichment-based metagenomic approaches, a more comprehensive understanding of microbial community structure in hypersaline environments can be achieved.

India↗
PubMed2006

Crystal structure of a glycosylated Fab from an IgM cryoglobulin with properties of a natural proteolytic antibody.

The 2.6 A (1 A=0.1 nm) resolution structure has been determined for the glycosylated Fab (fragment antigen binding) of an IgM (Yvo) obtained from a subject with Waldenström's macroglobulinaemia. Dynamic light scattering was used to estimate the gel point and monitor the formation of an ordered hydroscopic gel of Yvo IgM upon cooling. If a cryoglobulin forms gels in peripheral tissues and organs, the associated swelling and damage to microvasculature can result in considerable morbidity and mortality. The three-dimensional structure of the branched N-linked oligosaccharide associated with the CH1 domain (first constant domain of heavy chain) is reported. The carbohydrate may act to shield part of the lateral surface of the CH1 domain and crowd the junction between the CH1 and CH2 domains, thereby limiting the segmental flexibility of the Fab arms in intact Yvo IgM, especially at low temperatures. Recently, Yvo IgM was shown to have the properties of a naturally occurring proteolytic antibody [Paul, Karle, Planque, Taguchi, Salas, Nishiyama, Handy, Hunter, Edmundson and Hanson (2004) J. Biol. Chem. 279, 39611-39619; Planque, Bangale, Song, Karle, Taguchi, Poindexter, Bick, Edmundson, Nishiyama and Paul (2004) J. Biol Chem. 279, 14024-14032]. The Yvo protein displayed the ability to cleave, by a nucleophilic mechanism, the amide bonds of a variety of serine protease substrates and the gp120 coat protein of HIV. An atypical serine, arginine and glutamate motif is located in the middle of the Yvo antigen-binding site and displays an overall geometry that mimics the classical serine, histidine and aspartate catalytic triad of serine proteases. Our present findings indicate that pre-existing or natural antibodies can utilize at least one novel strategy for the cleavage of peptide bonds.

Amino Acid Sequence↗
PubMed2006

A common site of the Fc receptor gamma subunit interacts with the unrelated immunoreceptors FcalphaRI and FcepsilonRI.

The transmembrane (TM) region of the Fc receptor-gamma (FcRgamma) chain is responsible for the association of this ubiquitous signal transduction subunit with many immunoreceptor ligand binding chains, making FcRgamma key to a number of leukocyte activities in immunity and disease. Some receptors contain a TM arginine residue that interacts with Asp-11 of the FcRgamma subunit, but otherwise the molecular basis for the FcRgamma subunit interactions is largely unknown. This study reports residues in the TM region of the FcRgamma subunit are important for association with the high affinity IgE receptor FcepsilonRI and a leukocyte receptor cluster member, the IgA receptor FcalphaRI. FcRgamma residue Leu-21 was essential for surface expression of FcepsilonRIalpha/gamma2 and Tyr-8, Leu-14, and Phe-15 contributed to expression. Likewise, detergent-stable FcRgamma association with FcalphaRI was also dependent on Leu-14 and Leu-21 and in addition required residues Tyr-17, Tyr-25, and Cys-26. Modeling the TM regions of the FcRgamma dimer indicated these residues interacting with both FcalphaRI and FcepsilonRI are near the interface between the two FcRgamma TM helices. Furthermore, the FcRgamma residues interacting with FcalphaRI form a leucine zipper-like interface with mutagenesis confirming a complementary interface comprising FcalphaRI residues Leu-217, Leu-220, and Leu-224. The dependence of these two nonhomologous receptor interactions on FcRgamma Leu-14 and Leu-21 suggests that all the associated Fc receptors and the activating leukocyte receptor cluster members interact with this one site. Taken together these data provide a molecular basis for understanding how disparate receptor families assemble with the FcRgamma subunit.

Amino Acid Sequence↗
PubMed2006

The antibody repertoire in evolution: chance, selection, and continuity.

All jawed vertebrates contain the genetic elements essential for the function of the adaptive/combinatorial immune response, have diverse sets of natural antibodies resulting from segmental gene recombination, express comparable functional repertoires and can produce specific antibodies following appropriate immunization. Profound variability occurs in the third hypervariable (CDR3) segments of light and heavy chains even within antibodies of the same ostensible specificity. Germline VH and VL elements, as well as the joining (J) segments are highly conserved among the distinct vertebrate species. Conservation is particularly noted among the VH3-like sequences of all jawed vertebrates in the FR2 and FR3 segments, as well as in the FGXGT(R or K)L J-segment characteristic of light chains and TCRs and the WGXGT(uncharged)VT JH segments. Human VH3-53 and Vlambda6 family orthologs may be present over the entire range of vertebrates. Models of the three-dimensional structures of shark VH/VL combining sites indicate similarity in framework structure and comparable CDR usage to those of man. Although carcharhine shark VH regions show greater than 50% identity to the human VH germline prototype, searches of lower deuterostome and invertebrate databases fail to detect molecules with significant relatedness. Overall, antibodies of jawed vertebrates show tremendous individual diversity, but are constructed incorporating design features that arose with the evolutionary emergence of the jawed vertebrates and have been conserved through at least 450 million years of evolutionary time.

Amino Acid Sequence↗
PubMed2006

Enhanced major histocompatibility complex class I binding and immune responses through anchor modification of the non-canonical tumour-associated mucin 1-8 peptide.

Designing peptide-based vaccines for therapeutic applications in cancer immunotherapy requires detailed knowledge of the interactions between the antigenic peptide and major histocompatibility complex (MHC) in addition to that between the peptide-MHC complex and the T-cell receptor. Past efforts to immunize with high-affinity tumour-associated antigenic peptides have not been very immunogenic, which may be attributed to the lack of T cells to these peptides, having been deleted during thymic development. For this reason, low-to-medium affinity non-canonical peptides represent more suitable candidates. However, in addition to the difficulty in identifying such antigens, peptide binding to MHC, and hence its ability to induce a strong immune response, is limited. Therefore, to enhance binding to MHC and improve immune responses, anchor modifications of non-canonical tumour-associated peptides would be advantageous. In this study, the non-canonical tumour-associated peptide from MUC1, MUC1-8 (SAPDTRPA), was modified at the MHC anchor residues to SAPDFRPL (MUC1-8-5F8L) and showed enhanced binding to H-2Kb and improved immune responses. Furthermore, the crystal structure of MUC1-8-5F8L in complex with H-2Kb was determined and it revealed that binding of the peptide to MHC is similar to that of the canonical peptide OVA8 (SIINFEKL).

Animals↗
PubMed2005

Three-dimensional structures of carbohydrate determinants of Lewis system antigens: implications for effective antibody targeting of cancer.

Lewis system carbohydrate antigens have been shown to be expressed at high levels in many cancers of epithelial cell origin, including those of colon, breast, lung, prostate and ovary. The type 1 (Le(a) and Le(b)) antigens are important histo-blood groups, while type 2 (Le(x) and Le(y)) antigens in healthy individuals are only expressed, at relatively low levels, by a few tissues, including some epithelial cells. Thus, the type 2 antigens are considered to be tumour-associated antigens and are promising targets for cancer treatment, including antibody-based immunotherapy. In this review, we discuss the conformational characteristics of the free and bound forms of Lewis oligosaccharides and the 3D structures of antibodies in complex with Le(y) and Le(x) antigens. Collectively, the structural studies have demonstrated that the Lewis determinants are rigid structures, which generally maintain the same conformation in the free and bound states. The rigid nature and similarities in shape of type 1 and 2 Lewis oligosaccharides appear to make them perfectly suited to driving a structurally convergent immune response (at least in the case of Le(y) specific antibodies) toward a highly specific recognition of individual carbohydrate determinants, which is a goal in the development of effective antibody-based cancer treatments.

Animals↗
PubMed2004

Structural convergence of antibody binding of carbohydrate determinants in Lewis Y tumor antigens.

Antibodies targeting human epithelial carcinomas bearing Lewis Y (Le(y)) carbohydrate antigens provide a striking illustration of convergent immune recognition. We report a 1.9A resolution crystal structure of the Fab of a humanized antibody (hu3S193) in complex with the Le(y) tetrasaccharide, Fuc(alpha 1-->2)Gal(beta 1-->4)[Fuc(alpha 1-->3)]GlcNAc. Comparisons of the hu3S193 and BR96 antibodies bound to Le(y) tumor antigens revealed extremely similar mechanisms for recognition of the carbohydrate epitopes. Solvent plays a critical role in hu3S193 antibody binding to the Le(y) carbohydrate epitope. Specificity for Le(y) is maintained because a conserved pocket accepts an N-acetyl group of the core Gal(beta 1-->4)GlcNAc disaccharide. Closely related blood-group determinants (Le(a) and Le(b)) cannot enter the specificity pocket, making the Le(y) antibodies promising candidates for immunotherapy of epithelial cancer.

Amino Acid Sequence↗
PubMed2004

Three-dimensional structures of idiotypically related Fabs with intermediate and high affinity for fluorescein.

Multi-disciplinary studies of fluorescein-protein conjugates have led to the generation of a family of antibodies with common idiotypes and affinities for fluorescein ranging over five orders of magnitude. The high affinity 4-4-20 prototype traps the ligand in a highly complementary binding slot, which is lined by multiple aromatic side-chains. An antibody (9-40) of intermediate affinity belongs to the same idiotypic family as 4-4-20 and shares substantial amino acid identities within the VL and VH domains. To establish the structural basis for the affinity differences, we solved the crystal structure of the 9-40 Fab-fluorescein complex at a resolution of 2.3A. Similar to 4-4-20, 9-40 binds fluorescein in a tight aromatic slot with its xanthenonyl ring system accommodated by end-on insertion. However, the combined effects of the amino acid substitutions have resulted in reorganization of the binding site, with the HCDR3 loops showing the greatest differences in conformations. Access to the binding site of 9-40 is substantially more open, leaving the fluorescein's phenylcarboxylate moiety partially exposed to solvent. In addition to the usage of a different D (diversity) mini-gene encoding the HCDR3 loop, the decrease in fluorescein affinity in the 9-40 antibody family appears to be correlated with the substitution of histidine (9-40) for arginine (4-4-20) in position 34 of the antibody light chains.

Amino Acid Sequence↗
PubMed2004

Two monoclonal antibodies to precisely the same epitope of type II collagen select non-crossreactive phage clones by phage display: implications for autoimmunity and molecular mimicry.

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.

Amino Acid Motifs↗
PubMed2003

A glycopeptide in complex with MHC class I uses the GalNAc residue as an anchor.

Peptides bind MHC class I molecules by anchoring hydrophobic side chains into pockets in the peptide binding groove. Here, we report an immunogenic (in vitro and in vivo) MUC1 glycopeptide (MUC1-8-5GalNAc) bound to H-2Kb, fully crossreactive with the nonglycosylated variant. Molecular modeling showed that the central P5-Thr-GalNAc residue points into the C pocket and forms van der Waals and hydrogen bond interactions with the MHC class I. As predicted, GalNAc, a modified peptide carrying an additional anchor in the central C anchor pocket, increased the affinity by approximately 100-fold compared with the native low-affinity peptide (MUC1-8). The findings demonstrate that glycopeptides associated with MHC class I molecules can use GalNAc to anchor the peptide in the groove and enable high-affinity binding.

Acetylgalactosamine↗
PubMed2003

Structural analysis of IgG2A monoclonal antibodies in relation to complement deposition and renal immune complex deposition.

This study explores the structural features of murine monoclonal IgG2a anti-dinitrophenyl (DNP) antibodies that were previously shown to form immune complexes (IC) differing in their capacity to bind complement, their clearance from the circulation and their deposition in the kidney. Interestingly, the sequence of one of these antibodies has a missing stretch of 14 amino acids within FR3. Molecular modeling suggests that this sequence deletion corresponds to the loss of beta-pleated sheet structure for two beta-strands (designated 4-3 and 4-4) on the external surface of the V(H) domain. Despite this sequence and conformational abnormality, the antibody retains affinity for DNP comparable to other IgG2a antibodies. Data presented here identify monoclonal IgG2a antibodies that form IC with varying propensity for both complement binding and renal deposition and yet have similar V(H) domain sequences. In fact, in the case of two IgG2a antibodies that form IC with very different renal tropisms and complement binding capacity, sequence variation within V(H) was observed only at three clustered residues within FR2, a single residue within FR3 and nine clustered residues spanning CDR3 and FR4. Sequence and modeling analysis also yielded the paradoxical finding that an antibody forming IC with a relatively high capacity to serve as a target for complement binding displays a relatively low number of solvent exposed acceptor residues for C4b and C3b. These data underscore the complex relationship between V domain structure, complement activation and renal deposition of model IC.

Amino Acid Sequence↗
PubMed2003

Bovine IgM antibodies with exceptionally long complementarity-determining region 3 of the heavy chain share unique structural properties conferring restricted VH + Vlambda pairings.

Naturally occurring antibody repertoires of cattle (Bos taurus) include a group of IgMlambda antibodies with exceptionally long complementarity-determining region 3 of the heavy chain (CDR3H) segments, containing multiple Cys residues. These massive CDR3H segments will greatly influence the tertiary and quaternary structures of the bovine IgM combining sites. As an antibody's combining site is formed by both heavy and light chains, we have analyzed the nucleotide sequences and structural properties of the lambda-light chains that pair with micro -heavy chains containing exceptionally long CDR3H. There appears to be an exquisite selective pressure for the use of three V(lambda)1 genes (V(lambda)1x and two new V(lambda)1d and V(lambda)1e genes) in IgM with unusually long CDR3H. The V(lambda)1d and V(lambda)1e genes are similar to each other, but diverge from the other V(lambda)1 genes into two closely related subfamilies. The available bovine V(lambda) genes are classified into three V(lambda) gene families: V(lambda)1, V(lambda)2 and V(lambda)3 based on nucleotide similarity >/=80%. Further, analysis of total Ser content and positions of Ser residues in the sequences was found to be sufficient to classify the cattle V(lambda)1 subfamilies. Patterns of Ser residues differ for V(lambda) domains from ruminant species (e.g. cattle, sheep and goats) and other mammals (e.g. humans and mice). These 'Ser signatures' can be used to track divergent evolution in lambda-light chains. Interestingly, Ser90L in complementarity-determining region 3 of the light chain (CDR3L) occurred in all V(lambda) domains that pair with V(H) regions containing exceptionally long CDR3H. A structural role for Ser90L was revealed in homology models of V(lambda) domains, i.e. to hold the ascending polypeptide of CDR3L in a relatively tight space between the N-terminal segment and residues from CDR1L. The CDR3L of V(lambda) domains also occupied smaller volumes if paired to V(H) domains with extremely long CDR3H (>/=48 residues), and were more variable in their conformation and filled larger volumes if CDR3Hs were </=22 residues. Thus, the role of the lambda-light chains in these unusual cattle antibodies is probably to act as a relatively featureless supporting platform for the extremely long CDR3H regions, which undoubtedly are dominantly involved in binding to an antigen.

Amino Acid Sequence↗
PubMed2002

Three-dimensional structure of an immunoglobulin light-chain dimer with amyloidogenic properties.

The X-ray structure of an immunoglobulin light-chain dimer isolated from the urine as a "Bence-Jones protein" from a patient with multiple myeloma and amyloidosis (Sea) was determined at 1.94 A resolution and refined to R and R(free) factors of 0.22 and 0.25, respectively. This "amyloidogenic" protein crystallized in the orthorhombic P2(1)2(1)2(1) space group with unit-cell parameters a = 48.28, b = 83.32, c = 112.59 A as determined at 100 K. In the vital organs (heart and kidneys), the equivalent of the urinary protein produced fibrillar amyloid deposits which were fatal to the patient. Compared with the amyloidogenic Mcg light-chain dimer, the Sea protein was highly soluble in aqueous solutions and only crystallized at concentrations approaching 100 mg ml(-1). Both the Sea and Mcg proteins packed into crystals in highly ordered arrangements typical of strongly diffracting crystals of immunoglobulin fragments. Overall similarities and significant differences in the three-dimensional structures and crystalline properties are discussed for the Sea and Mcg Bence-Jones proteins, which together provide a generalized model of abnormalities present in lambda chains, facilitating a better understanding of amyloidosis of light-chain origin (AL).

Amino Acid Sequence↗
PubMed2002

Anti-sm autoantibodies in systemic lupus target highly basic surface structures of complexed spliceosomal autoantigens.

Autoantibodies directed against spliceosomal proteins are a common and specific feature of systemic lupus erythematosus. These autoantibodies target a collection of proteins, including Sm B, B', D1, D2, and D3. We define the common antigenic targets of Sm D2 and D3 and examine their role in spliceosomal autoimmunity. Our results define nine major common epitopes, five on Sm D2 and four on Sm D3. These epitopes have significantly higher (more basic) isoelectric points than do nonantigenic regions. In fact, this association is of sufficient power to make isoelectric point an excellent predictor of spliceosomal antigenicity. The crystallographic structure of Sm D2 and D3 is now partially described. The anti-Sm D2 and D3 antigenic targets are located on the surface of the respective three-dimensional complexed proteins, thereby suggesting that these epitopes are accessible in the native configuration. All but one of these nine epitopes conspicuously avoid the specific regions involved in intermolecular interactions within the spliceosomal complex. One of the D3 epitopes (RGRGRGMGR) has significant sequence homology with a major antigenic region of Sm D1 (containing a carboxyl-terminal glycine-arginine repeat), and anti-D3 Abs cross-react with this epitope of Sm D1. These results demonstrate that spliceosomal targets of autoimmunity are accessible on native structure surfaces and that cross-reactive epitopes, as well as structural associations of various spliceosomal Ags, may be involved in the induction of autoimmunity in systemic lupus.

Adolescent↗
PubMed2002

Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity.

Alpha(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Galalpha(1,3)Gal. This glycosyltransferase has been studied extensively because the Galalpha(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized. Comparison of amino acid sequences of GT from several species with the human A and B transferases suggest that His271 of pig GT may be critical for recognition of the donor substrate, UDP-Gal. Using pig GT as the representative member of the GT family, we show that replacement of His271 with Ala, Leu, or Gly caused complete loss of function, in contrast to replacement with Arg, another basic charged residue, which did not alter the ability of GT to produce Galalpha(1,3)Gal. Molecular modeling showed that His271 may interact directly with the Gal moiety of UDP-Gal, an interaction possibly retained by replacing His with Arg. However, replacing His271 with amino acids found in alpha(1,3)GalNAc transferases did not change the donor nucleotide specificity. Thus His271 is critical for enzymatic function of pig GT.

Amino Acid Sequence↗
PubMed2001

Crystallization and preliminary X-ray diffraction analysis of monkey dimeric dihydrodiol dehydrogenase.

Dihydrodiol dehydrogenase catalyzes the NADP(+)-linked oxidation of trans-dihydrodiols of aromatic hydrocarbons to corresponding catechols and exists in multiple forms in mammalian tissues. The dimeric form of mammalian dihydrodiol dehydrogenase has a primary structure distinct from the previously known mammalian enzymes and may constitute a novel protein family with the prokaryotic proteins. Monkey kidney dimeric dihydrodiol dehydrogenase was crystallized from buffered ammonium phosphate solution using the hanging-drop vapour-diffusion method. The crystals diffract to 2.65 A resolution in the laboratory and belong to the hexagonal P6(1)22 or P6(5)22 space group, with unit-cell parameters a = b = 122.8, c = 121.3 A, alpha = beta = 90, gamma = 120 degrees.

Animals↗
PubMedDate not supplied

Crystal structures of human antibodies: a detailed and unfinished tapestry of immunoglobulin gene products.

Sequencing of all human immunoglobulin (Ig) germline gene segments has recently been completed. However, our first glimpses of the recombined products of this combinatorial gene system were in the 1970s, in landmark publications, reporting the crystal structures of two human myeloma proteins, the Mcg lambda light chain dimer and the New IgG1(lambda) Fab. Although numerous crystal structures of murine and human antibodies have now been determined, only a relatively small proportion of the human germline genes have had their corresponding protein three-dimensional structures resolved. Therefore, further structural investigations are required before the inherent diversity of the antibody repertoire can be fully appreciated. We discuss the detailed structural information available for human antibodies with regard to their immune functions. Also discussed, is how the structural information is finding application in the 'humanization' of murine antibodies as part of their development as 'biopharmaceuticals' for the treatment of human disease.

Animals↗