Phillip W. Berman

Delineation of a region of the human immunodeficiency virus type 1 gp120 glycoprotein critical for interaction with the CD4 receptor

The primary event in the infection of cells by HIV is the interaction between the viral envelope glycoprotein, gp120, and its cellular receptor, CD4. A recombinant form of gp120 was found to bind to a recombinant CD4 antigen with high affinity. Two gp120-specific murine monoclonal antibodies were able to block the interaction between gp120 and CD4. The gp120 epitope of one of these antibodies was isolated by immunoaffinity chromatography of acid-cleaved gp120 and shown to be contained within amino acids 397-439. Using in vitro mutagenesis, we have found that deletion of 12 amino acids from this region of gp120 leads to a complete loss of binding. In addition, a single amino acid substitution in this region results in significantly decreased binding, suggesting that sequences within this region are directly involved in the binding of gp120 to the CD4 receptor.

Engineering Chinese hamster ovary cells to maximize sialic acid content of recombinant glycoproteins.

We have engineered two Chinese hamster ovary cell lines secreting different recombinant glycoproteins to express high levels of human β1,4-galactosyltransferase (GT, E.C. 2.4.1.38) and/or α2,3-sialyltransferase (ST, E.C. 2.4.99.6). N-linked oligosaccharide structures synthesized by cells overexpressing the glycosyltransferases showed greater homogeneity compared with control cell lines. When GT was overexpressed, oligosaccharides terminating with GlcNAc were significantly reduced compared with controls, whereas overexpression of ST resulted in sialylation of ≥90% of available branches. As expected, GT overexpression resulted in reduction of oligosaccharides terminating with GlcNAc, whereas overexpression of ST resulted in sialylation of ≥90% of available branches. The more highly sialylated glycoproteins had a significantly longer mean residence time in a rabbit model of pharmacokinetics. These experiments demonstrate the feasibility of genetically engineering cell lines to produce therapeutics with desired glycosylation patterns.

Sequence analysis, cellular localization, and expression of a neuroretina adhesion and cell survival molecule

A cDNA for purpurin, a secreted 20,000 dalton neural retina cell adhesion and survival protein, has been sequenced and expressed in mammalian cells. Purpurin mRNA is found in both embryonic and adult retina, but not the brain, heart, or liver. The protein is highly concentrated in the neural retina between the pigmented epithelium and the outer segments of the photoreceptor cells; it is synthesized by photoreceptor cells. The predicted purpurin sequence contains 196 residues, has approximately 50% sequence homology with serum retinol binding protein, and is a member of the alpha-2 mu-globulin superfamily. Purpurin binds retinol and may play a major role in retinol transport across the interphotoreceptor cell matrix.

Engineering glycoproteins for use as pharmaceuticals

Abstract The fact that glycosylation is not a significant process in prokaryotes means that many of the proteins produced by genetically engineered bacteria are not identical to their eukaryotic counterparts. Although glycosylation affects the physical, chemical and biological nature of proteins, its pharmacological value in potential protein pharmaceuticals is not easy to predict. However, the development of mammalian cell culture methods for expressing recombinant DNA-derived glycoproteins will permit further studies in the field.

The cytoplasmic tail of HIV-1 gp160 contains regions that associate with cellular membranes

The HIV-1 envelope glycoprotein gp160 associates with cellular membranes via a discrete transmembrane domain. Unlike other retroviral envelope proteins, however, gp160 also forms a secondary association with the lipid bilayer mediated by one or more regions located in the cytoplasmic tail. We have expressed the full cytoplasmic tail sequence of gp160, as a fusion protein with the HSV-1 glycoprotein D signal sequence, transiently in a human embryonic kidney cell line. Our results show that in the absence of any defined transmembrane domain or stop transfer sequence, the protein corresponding to the cytoplasmic tail of HIV-1 gp160 formed stable interactions with cellular membranes that mediated its export to the cell surface.

Secretion of glycosylation site mutants can be rescued by the signal/pro sequence of tissue plasminogen activator

Strategies that prevent the attachment of N‐linked carbohydrates to nascent glycoproteins often impair intracellular transport and secretion. In the present study, we describe a method to rescue the intracellular transport and secretion of glycoproteins mutagenized to delete N‐linked glycosylation sites. Site‐directed mutagenesis was used to delete N‐linked glycosylation sites from a chimeric protein, TNFR‐IgG1. Deletion of any of the three glycosylation sites in the TNFR portion of the molecule, alone or in combination, resulted in a moderate or near total blockade of TNFR‐IgG1 intracellular transport and secretion. Pulse chase experiments suggested that the glycosylation site mutants accumulated in the endoplasmic reticulum (ER) and were inefficiently exported to the Golgi apparatus (GA). Replacement of the TNFR signal sequence with the signal/pro sequence of human tissue plasminogen activator (tPA) overcame the blockade to intracellular transport, and restored secretion to levels comparable to those achieved with the fully glycosylated molecule. Ligand binding studies suggested that the secreted glycosylation variants possessed binding characteristics similar to the fully glycosylated protein. This study demonstrates that N‐terminal sequences of tPA are unexpectedly efficient in facilitating transport from the ER to the GA and suggests that these sequences contain a previously unrecognized structural element that promotes intracellular transport. J. Cell. Biochem. 75:446–461, 1999.

Prospects for vaccines for herpes simplex viruses

A subunit vaccine consisting of glycoprotein D from the surface of herpes simplex virus was prepared using recombinant deoxyribonucleic acid techniques to transfer the viral gene for glycoprotein D into Chinese hamster ovary cells. The glycoprotein D produced by these Chinese hamster cells combined with appropriate adjuvants effectively immunized mice and guinea pigs against herpes simplex infections. In addition to protecting guinea pigs from serious or fatal disease, the vaccine prevented a small group of guinea pigs from developing latent ganglionic infections. The vaccine has not yet been used in humans.

Production of Viral Glycoproteins in Genetically Engineered Mammalian Cell Lines for Use as Vaccines against Herpes Simplex Virus and the Acquired Immune Deficiency Syndrome Retrovirus

One promise of recombinant DNA technology is the possibility of developing new and improved vaccines to prevent the transmission of infectious diseases. Using these techniques, it is possible to produce virtually unlimited quantities of the surface antigens of pathogenic organisms without resorting to the large-scale culture of the pathogen itself. Conventional methods of vaccine production (i.e., live attenuated viruses, killed viruses, or extracts of killed viruses) are limited by the fact that some pathogens are difficult or uneconomical to grow. In addition, society is still haunted by fears that such vaccine preparations could be contaminated by unattenuated or inadequately inactivated virus. Even when the most stringent production methods are applied, manufacturers must be concerned with the possibility that a vaccine could induce latent infections, oncogenic transformation, or autoimmunity. The recombinant DNA approach to vaccine development circumvents these problems by providing a preparation consisting of a single highly purified protein, derived from a safe noninfectious source.