Teresa I. Mitchell

Humanization of Yeast to Produce Complex Terminally Sialylated Glycoproteins

Yeast is a widely used recombinant protein expression system. We expanded its utility by engineering the yeast Pichia pastoris to secrete human glycoproteins with fully complex terminally sialylated N-glycans. After the knockout of four genes to eliminate yeast-specific glycosylation, we introduced 14 heterologous genes, allowing us to replicate the sequential steps of human glycosylation. The reported cell lines produce complex glycoproteins with greater than 90% terminal sialylation. Finally, to demonstrate the utility of these yeast strains, functional recombinant erythropoietin was produced.

A Novel Host/Tumor Cell Interaction Activates Matrix Metalloproteinase 1 and Mediates Invasion through Type I Collagen

Along with degradation of type IV collagen in basement membrane, destruction of the stromal collagens, types I and III, is an essential step in the invasive/metastatic behavior of tumor cells, and it is mediated, at least in part, by interstitial collagenase 1 (matrix metalloproteinase 1 (MMP-1)). Because A2058 melanoma cells produce substantial quantities of MMP-1, we used these cells as models for studying invasion of type I collagen. With a sensitive and quantitative in vitro invasion assay, we monitored the ability of these cells to invade a matrix of type I collagen and the ability of a serine proteinase inhibitor and all-trans-retinoic acid to block invasion. Although these cells produce copious amounts of MMP-1, they do not invade collagen unless they are co-cultured with fibroblasts or with conditioned medium derived from fibroblasts. Our studies indicate that a proteolytic cascade that depends on stromal/tumor cell interactions facilitates the ability of A2058 melanoma cells to invade a matrix of type I collagen. This cascade activates latent MMP-1 and involves both serine proteinases and MMPs, particularly stromelysin 1 (MMP-3). All-trans-retinoic acid (10−6 m) suppresses the invasion of tumor cells by several mechanisms that include suppression of MMP synthesis and an increase in levels of tissue inhibitor of metalloproteinases 1 and 2. We conclude that invasion of stromal collagen by A2058 melanoma cells is mediated by a novel host/tumor cell interaction in which a proteolytic cascade culminates in the activation of pro-MMP-1 and tumor cell invasion.

Transforming growth factor β inhibitory element in the rabbit matrix metalloproteinase-1 (collagenase-1) gene functions as a repressor of constitutive transcription

Transforming growth factor beta (TGF-beta) is a potent modulator of the extracellular matrix, enhancing collagen synthesis and regulating expression of several genes that encode the matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix. In this study, we explored the mechanisms whereby TGF-beta inhibits expression of the MMP-1 (collagenase 1) gene. We used transient transfection and gel mobility shift assays to characterize a TGF beta inhibitory element (TIE) at -249 bp in the rabbit MMP-1 promoter, which is also conserved at -246 bp in the human gene. This sequence shares homology to a previously identified TIE in the rat stromelysin-1 (MMP-3) promoter, where it is located at -709 bp. Mutational analyses and transient transfections indicate that MMP-1 TIE functions both as a constitutive repressor of MMP-1 gene expression and, in the presence of TGF-beta, as an antagonist of transcriptional induction by phorbol esters. c-Fos binds to the TIE in the rabbit MMP-1 promoter, along with other nuclear proteins, even in the absence of treatment with TGF-beta. However, the pattern of proteins binding to the TIE is altered in the presence of nuclear extracts from TGF-beta-treated cells, suggesting that TGF-beta leads to an alteration in protein/DNA interaction, with subsequent modulation of MMP-1 gene expression. We conclude that in the rabbit MMP-1 promoter, the TIE has dual functions as a repressor of basal transcription and as a mediator of the biologic effects of TGF-beta. Furthermore, these dual functions provide additional and subtle mechanisms for regulating MMP-1 gene expression under a variety of biological and pathological conditions.

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study.

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in a glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia–produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependant cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.

The acute phase reactant serum amyloid A (SAA3) is a novel substrate for degradation by the metalloproteinases collagenase and stromelysin

We found that the matrix metalloproteinases collagenase (MMP-1) and stromelysin (MMP-3) each has the ability to degrade a novel substrate, serum amyloid A (SAA3). SAA3 is a product of rabbit synovial fibroblasts stimulated with phorbol esters or interleukin-1, and it acts in an autocrine or paracrine manner to induce collagenase in both rabbit and human fibroblasts. Recombinant rabbit fibroblast procollagenase and human fibroblast prostromelysin were produced by baby hamster kidney (BHK) cells stably transfected with these genes, and latent enzyme was activated with aminophenylmercuric acetate (APMA). The Km for both enzymes was approximately 10 microM, and the Vmax for collagenase was approximately 6 pmol/minute/100 ng enzyme, while that for stromelysin was about 3-fold faster. Treatment of SAA3 with either enzyme generated a fragment of approx. 6 kDa that has the same amino terminus as the parent molecule, but this fragment was rapidly degraded. We have been unable to isolate C-terminal fragments, suggesting that the mature protein is cleaved at multiple sites and/or that the initial cleavage fragment is readily digested. The amino acid composition of the 6 kDa fragment suggests that the 14 kDa protein is cleaved at residues 50-57, a hydrophobic region that is conserved between rabbit SAA3 and human SAA1. We conclude that the ability of collagenase and stromelysin to degrade SAA3 broadens the repertoire of substrates for these matrix degrading enzymes, and we speculate that the presence of a feedback mechanism that can subvert the autocrine/paracrine stimulation of matrix-degrading enzymes may play a role in limiting matrix degradation during inflammatory conditions.

A combinatorial genetic library approach to target heterologous glycosylation enzymes to the endoplasmic reticulum or the Golgi apparatus of Pichia pastoris

To humanize the glycosylation pathway in the yeast Pichia pastoris, we developed several combinatorial genetic libraries and used them to properly localize active eukaryotic mannosidases and sugar transferases. Here we report the details of the fusion of up to 66 N‐terminal targeting sequences of fungal type II membrane proteins to 33 catalytic domains of heterologous glycosylation enzymes. We show that while it is difficult to predict which leader/catalytic domain will result in the desired activity, analysis of the fusion protein libraries allows for the selection of the leader/catalytic domain combinations that function properly. This combinatorial approach, together with a high‐throughput screening protocol, has allowed us to humanize the yeast glycosylation pathway to secrete human glycoprotein with complex N‐glycosylation. Copyright

Retinoid‐Mediated Suppression of Tumor Invasion and Matrix Metalloproteinase Synthesis

ABSTRACT: Cancer mortality usually results from the tumor invading the local environment and metastasizing to vital organs, e.g. liver, lung, and brain. Degradation of the extracellular matrix is, therefore, the sine qua non of tumor cell invasion. this degradation is mediated mainly by MMPs, and thus, inhibition of MMP synthesis is a target for anticancer agents. Tumor cells must traverse both the basement membrane (type IV collagen) and the interstitial stroma (type I collagen). Therefore, we used scanning electron microscopy to examine the invasive behavior of several aggressive tumor cell lines, A2058 melanoma cells, and SCC and FaDu squamous cell carcinomas through these matrices; and we monitored the ability of all‐trans retinoic acid and several RAR‐specific ligands to block invasion. We demonstrate that several retinoids, which are specific RAR α, β, or γ agonists/antagonists, selectively inhibited MMP synthesis in the three tumor cell lines. However, there was not a common pattern of MMP inhibition by a particular retinoid. For instance, a RAR α antagonist suppressed MMP‐1 and MMP‐2 synthesis in the melanoma cell line, but not in the FaDu or SCC‐25 cells. On the other hand, synthesis of MMP‐1 and MMP‐9 by the FaDu cells was affected hardly at all, while a RARγ antagonist reduced the levels of MMP‐2. Only all‐trans retinoic acid reduced MMP‐1 synthesis in these cells. We postulate that the differences may be related to a differential pattern of RAR expression in each of these cells, and that the RARs expressed by each cell line may not be targets of these RAR specific compounds. All‐trans retinoic acid is a pan ligand, binding to all three RARs and, therefore, may modulate gene expression more generally. We conclude that the power of these new ligands lies in their specificity, which can be directed towards modulating expression of certain RARs and, thus, of certain MMPs. By blocking MMP synthesis, retinoids may be effective in cancer therapy by decreasing tumor invasiveness.

Characteristics of a precursor to vasopressin-associated bovine neurophysin.

The neurophysin produced by vasopressinergic neurons of the ox has been called vasopressinassociated bovine neurophysin (VP-BNP) [l] and it has long been suspected that such neurophysins are initially translated as part of the prohormone structure of vasopressin [2,3,4; for reviews see 5,6]. Some elegant work was recently performed to obtain the sequence of a cDNA encoding both vasopressin and its neurophysin from bovine hypothalamus [7]. This provided proof that both molecules arise as part of the same precursor. The primary structure of this precursor derived from a knowledge of the nucleotide sequence of an mRNA, places vasopressin on the N-terminal side of VP-BNP and joined to it by the tripeptide sequence, -Gly-Lys-Arg-. A 39-residue glycopeptide is bridged to the C-terminus of VP-BNP by an Arg. On the N-terminal side of vasopressin there is a 19-residue peptide believed to represent the leader sequence of the translated prohormone. The prohormone (possibly minus the leader sequence) is believed to be processed within neurosecretory granules during transport from the perikarya of neurons to axonal storage sites in the neural lobe from where the products of intragranular proteolysis, vasopressin and VP-BNP, are released [5,6]. We have earlier demonstrated the presence in neurosecretory granules of an enzyme which might be involved in such a transformation [8]. Here we describe the characteristics of precursors to VPBNP which were isolated from bovine neural lobes. These proteins provide us with a partial confirmation of the structure derived for the prohormone from nucleotide sequencing, and also provide information concerning the manner in which processing from prohormone to products might occur within the neurosecretory granules.

Evolution of neurophysin proteins: partial amino acid sequences of rat neurophysins

Neurophysins (NPs) are proteins elaborated by cells of the hypothalamo-neurohypophysial system, and within this system there is a close association between the biosynthesis of each neurophysin and the biosynthesis of each neurohypophysial hormone [ 1,2]. This lead to the proposal of a classification based on this association [3-S]. In the rat, there are 3 major forms of these proteins which were formerly named rat neurophysins I, II and III, according to their electrophoretic mobility at pH 9.5 [6,7]. Rat neurophysin I has been renamed vasopressin-associated neurophysin (VP-RNP), and rat neurophysin II renamed oxytocin-associated neurophysin (OT-RNP) [ 1,3]. Rat neurophysin III was found [8,9] to be a metabolic derivative of OT-RNP, and is now referred to as OT-RNP’ [2]. The close relationship between one neurophysin and one hormone is perhaps best exemplified by Brattleboro homozygotes, rats with hereditary diabetes insipidus [lo]. These rats have an apparent inability to synthesize vasopressin and VP-RNP (RNP I), while their synthesis of oxytocin, OT-RNP and OT-RNP’ is unimpaired [ 1,2,6]. The view now generally accepted is that each neurophysin and its hormone initially arise as parts of a common precursor molecule [ 1,2,11]. Amino acid sequences have been described for neurophysins of the ox [i2,13], the pig [14,15], and the sheep [ 161 (mammals of the order Artiodactyla); for one neurophysin of the horse [ 171 (order Perissodactyla); and for one neurophysin of the whale [ 181 (order Cetacea). Also, the amino acid sequence of the N-terminal 54-residues of oxytocin-associated human neurophysin has also been determined [4,19] (order Primates). The N-terminal 30-residues of rat neurophysin I and rat neurophysin II (order Rodentia) have been identified in [ 201.

N-linked glycan characterization of heterologous proteins.

Our laboratory has focused on the re-engineered of the secretory pathway of Pichia pastoris to perform glycosylation reactions that mimic processing of N-glycans in humans and other higher mammals (1,2). A reporter protein with a single N-linked glycosylation site, a His-tagged Kringle 3 domain of human plasminogen (K3), was used to identify combinations of optimal leader/catalytic domain(s) to recreate human N-glycan processing in the Pichia system. In this chapter we describe detailed protocols for high-throughput purification of K3, enzymatic release of N-glycans, matrix-assisted laser desorption ionization time-of-flight and high-performance liquid chromatography analysis of the released N-glycans. The developed protocols can be adapted to the characterization of N-glycans from any purified protein expressed in P. pastoris.