The primary focus of the Pierce laboratory centers on understanding the regulation of intercellular recognition and adhesion, particularly those events that involve protein-oligosaccharide interactions. Almost 20 years ago, observations were made that when vertebrate cells became oncogenically transformed, a highly reproducible but poorly understood change in their cell surface oligosaccharides takes place. This change was observed as a shift toward higher molecular weight when the oligosaccharides were separated using gel permeation chromatography. This chromatographic shift was observed for a large number of cell types and for diverse transforming agents, from mutagens to tumor virus infection. The first focus of the Pierce laboratory became to identify the molecular mechanism that causes this change in oligosaccharide expression and to understand how the alteration may regulate cell-cell and cell-substrate adhesion.
The system Dr. Pierce chose to investigate in detail was the transformation of BHK fibroblasts by the Rous sarcoma virus through its src oncogene. The group first developed analytical techniques that allowed them to demonstrate that the oligosaccharide change caused by src transformation was a specific increase in a particular N-linked oligosaccharide structure, referred to as the b(1,6) branch. With a collaborator in Canada Pierce developed a novel system utilizing a synthetic modified trisaccharide to assay for the enzyme that synthesizes this oligosaccharide branch, N-acetylglucosaminyltransferase V or GlcNAc-T V. They showed that the activity of this enzyme alone increased 6-fold after src transformation of the BHK cells. As a consequence of these studies, Pierce received an American Cancer Society Faculty Research Award, which allowed the group to concentrate on understanding how src caused a specific increase in GlcNAc-T V activity and the functional consequences of subsequent cell-surface oligosaccharide alterations.
After several years of intensive work, Pierce and his group purified GlcNAc-T V a million-fold from rat kidney, and after he moved his laboratory to the University of Georgia, the group was able to obtain the peptide sequence. Aided by PCR techniques, they were finally able to isolate a full length cDNA which encoded the enzyme, greatly aided by their collaboration with Dr. N. Fregien of the University of Miami Medical School. They have also produced large quantities of recombinant enzyme for their studies on the physical structure of GlcNAc-T V and ongoing attempts to crystallize the enzyme for X-ray diffraction studies. Since obtaining the cDNA, the group's focus has been to understand the mechanism of the src-induced increases in GlcNAc-T V catalytic activity. They have now succeeded in demonstrating the signaling pathway by which src causes a 7-fold increase in promoter activity of the GlcNAc-T V gene and consequential increase in mRNA levels. They have also identified a small segment of genomic DNA upstream from the promoter that contains sequences known to bind particular enhancers of transcription. Their present focus is to understand the details of the regulation of the GlcNAc-T V gene, since its expression also appears to be regulated in some cell types by translational control. They have recently shown that an oncogene often amplified in breast cancer, neu, causes a specific increase in GlcNAc-T V activity and alters cell-surface glycoprotein glycosylation.
The other focus of the Pierce group's work is on determining the functional consequences of the increase in b(1,6) branched oligosaccharides on particular glycoproteins on the cell surface. These studies are critical because results from several laboratories have demonstrated that alteration of the levels of these oligosaccharides on the surface of tumor cells can regulate their invasiveness and metastatic potential. For example, a clinical study demonstrated increased GlcNAc-T V product on cells from the majority of patients with breast cancer, and the increase correlated well with disease progression. The hypothesis that the Pierce group is attempting to test in these functional studies is that GlcNAc-T V expression can regulate cellular adhesion properties. They are presently collaborating with two laboratories to construct transgenic mice that do not express GlcNAc-T V and mice that over-express the enzyme in specific tissues, including the mammary gland. Over-expression of GlcNAc-T V in two different cell types stimulates cell migration and decreases intercellular adhesion, suggesting that this step is a necessary but not sufficient step in the progression of proliferating cells to a malignant phenotype.
In a related study, the group is identifying the glycoproteins that show altered glycosylation in human breast carcinomas. This knowledge will then be used to develop monoclonal antibodies to these altered glycoproteins as potential reagents for a serum-based assay for the presence of breast carcinoma.
Recently, in collaboration with the Moremen group at the CCRC, Pierce's group has discovered a new family of vertebrate carbohydrate-binding proteins (lectins). The first member of this family that they cloned is present in the cortical granules of the unfertilized oocyte of the frog Xenopus laevis. This lectin is released extracellularly at fertilization, where it binds oligosaccharides on glycoproteins in the egg jelly surrounding each egg. This reaction cross-links the glycoproteins, forming part of the fertilization membrane that blocks polyspermy. The group has shown that the lectin is also expressed after fertilization during the time that embryonic organs are forming. The lectin is expressed and secreted from specific cell types undergoing active cell migration and cell adhesion events. They have now shown that homologues of this lectin are expressed in adult human and mouse tissues; the highest levels are found in heart muscle, small intestine, colon, and other mesodermally derived cell types. They have recently identified another member of this lectin family that is expressed specifically only in small intestine, and there are indications that other members of this family are yet to be identified in vertebrate tissues. Both lectins are present only in endothelial cells, and their chromosomal localization, 1q23, is in the identical position as other endothelial adhesion lectins, including the selectins. The project's current focus is to identify the cellular targets and oligosaccharide ligands for the lectins, which they have termed "Endothelial Lectins 1 and 2", and determine what hormones or other stimuli regulate their biosynthesis and release. Additional experiments in other laboratories around the world suggest that the endothelial lectins and the Xenopus oocyte lectin are members of a new superfamily of lectins that has members in fungi and animals.
Potential commercial applications of Dr. Pierce's research include the detection of tumor antigens, inhibiting metastasis-associated enzymes, and engineering animal cells to secrete recombinant proteins with defined, homogeneous oligosaccharide structures. Dr. Pierce's work is supported by the National Institutes of Health.