Pedro A. Prieto

Glycoconjugates in human and transgenic animal milk.

Human milk samples contain a particularly rich collection of oligosaccharides compared with other milk samples. The synthesis of these molecules should depend on the expression of glycosyltransferases and the presence of sugar nucleotides in lactating mammary glands. We set out to produce transgenic animals expressing glycosyltransferases during lactation with the purpose of exploring the following issues: a) Is it possible to synthesize human milk oligosaccharides in lactating mammary glands of nonhuman animals?, b) Is it possible to express during lactation homologous, tissue specific glycosyltransferases that are not normally expressed in lactating mammary tissue?, and c) What is the effect of expressing a human glycosyltransferase in different animal species? Simultaneously, we embarked on a research program to study short-chain neutral human milk oligosaccharides--no larger than hexasaccharides--to understand the natural variation of milk sugars and glycoproteins. The reagents and methods developed to study human milk oligosaccharides and glycoproteins were also applied to the study of milk from transgenic animals. Our results indicate that mice predictably express transgene-encoded glycosyltransferases and their secondary gene products, oligosaccharides and remodeled glycoproteins. This was true even when the transgene encoded a homologous galactosyltransferase. Also, it was possible to synthesize fucosylated glycoconjugates in mouse milk using two different fucosyltransferases, thus demonstrating that is feasible to emulate the synthesis occurring in the human lactating mammary gland. Experiments with transgenic rabbits yielded different phenotypes, some of them unexpected. Taken together, our results answer the questions stated above but open even more intriguing areas of inquiry.

A new sialyloligosaccharide from human milk: isolation and characterization using anti-oligosaccharide antibodies.

A previously undescribed sialyloligosaccharide has been isolated from human milk using a specific anti-sialyloligosaccharide antibody. Structural studies of the radiolabeled oligosaccharide by enzyme degradation and binding by specific anti-oligosaccharide sera are consistent with the following structure: (sequence in text) The oligosaccharide is present only in milk from donors who secrete A, B, or H blood group substances; this is consistent with the requirement of at least one copy of the Se (Secretor) gene necessary for the synthesis of oligosaccharides with Fuc alpha 1-2Gal . . . linkages.

A new ganglioside in human meconium detected with antiserum against human milk sialyltetrasaccharide a

Antiserum directed against the alditol derivative of the human milk monosialyloligosaccharide sialyltetrasaccharide a [D. F. Smith, P. A. Prieto, and B. V. Torres (1985) Arch. Biochem. Biophys. 241, 298-303] is used to detect a new ganglioside in human meconium by direct binding on nitrocellulose filters of the sialyl[3H]oligosaccharide alditol obtained from gangliosides after ozonolysis and alkali fragmentation. The sialyl[3H]oligosaccharide is purified by affinity chromatography on a column containing anti-sialyltetrasaccharide a antibodies. The affinity-purified sialyl[3H]oligosaccharide cochromatographs with the 3H-labeled alditol derivative of authentic sialyltetrasaccharide a from human milk. Results of sequential enzyme degradation of the pure sialyl[3H]oligosaccharide and cochromatography of the digestion products with standards are consistent with the presence in meconium of a monosialylganglioside with the structure NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc-ceramide. This ganglioside is presumably the biosynthetic precursor of the sialyl-Lea ganglioside [G. C. Hansson and D. Zopf (1985) J. Biol. Chem. 260, 9388-9392], which is also a component of human meconium.

Special Considerations for Glycolipids and Their Purification

This unit describes the antigenic stimulation of in vitro antibody production by B cells and the subsequent measurement of secreted antibodies. A generalized system for inducing in vitro antibody production is presented along with a procedure for quantifying the number of antibody‐producing cells by plaque‐forming cell (PFC) assays: the Cunningham‐Szenberg technique and the Jerne‐Nordin technique. The assay can be modified as described to measure all classes of antibodies or to enumerate total immunoglobulin‐secreting B cells. A protocol for preparing the resting B cells by Percoll gradient centrifugation is also described.

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcOH)☆

Abstract The sialyloligosaccharide, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LS-tetrasaccharide a ), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAcα2-3Galβ1-3[NeuAcα2-6]GlcNAcβ1-3Galβ1-4Glc (disialyllacto- N -tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3 H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding ( K a > 10 6 m −1 ) requires sialic acid linked α2–3 to the nonreducing terminal galactose residue of reduced lacto- N -tetraose (Galβ1-3GlcNAcβ1-3Galβ1-4Glc OH ). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Galβ1-3[NeuAcα2-6]GlcNAcβ1-3Galβ1-4Glc) and LS-tetrasaccharide c (NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [ D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255 , 55–59 ].

Transgenic animals and nutrition research

Transgenic animals are useful tools for the study of biological functions of proteins and secondary gene products synthesized by the action of protein catalysts. Research in nutrition and allied fields is benefiting from their use as models to contrast normal and altered metabolism. Although food, nutritional products, and ingredients from transgenic animals have not yet reached consumers, the technologies for their production are maturing and yielding exciting results in experimental and farm animals. Regulatory governmental bodies are already issuing guidelines and legislation in anticipation of the advent of these products and ingredients. This review summarizes available technology for the production of transgenic animals, discusses their scientific and commercial potential, and examines ancillary issues relevant to the field of nutrition.