Siv Annegrethe Hjorth

One-step purification and characterization of human pancreatic lipase expressed in insect cells

A cDNA clone encoding the sequence of human pancreatic lipase (HPL) was subcloned into the baculovirus transfer vector pVL1392 and used in co‐transfection of Spodoptera frugiperda (Sf9) insect cells with wild‐type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. A single recombinant protein (50 kDa) secreted by Sf9 cells was detectable in the culture medium 24 h post‐infection using both anti‐HPL polyclonal antibodies and potentiometric measurements of lipolytic activity. The expression level reached 40 mg/l of enzyme at 6 days. A single cation‐exchange chromatography was sufficient to obtain a highly pure recombinant HPL as demonstrated by N‐terminal sequencing, amino acid composition and carbohydrate analysis, as well as by mass spectrometry. These analyses revealed the production of mature protein with the correct processing of signal peptide and an homogenous glycosylation pattern. The kinetic properties of recombinant and native HPL were compared. Both enzymes showed similar profiles of interfacial activation, inhibition by bile salts and re‐activation by colipase.

Cutting Edge: Identification of Neutrophil PGLYRP1 as a Ligand for TREM-1

Triggering receptor expressed on myeloid cells (TREM)-1 is an orphan receptor implicated in innate immune activation. Inhibition of TREM-1 reduces sepsis in mouse models, suggesting a role for it in immune responses triggered by bacteria. However, the absence of an identified ligand has hampered a full understanding of TREM-1 function. We identified complexes between peptidoglycan recognition protein 1 (PGLYRP1) and bacterially derived peptidoglycan that constitute a potent ligand capable of binding TREM-1 and inducing known TREM-1 functions. Interestingly, multimerization of PGLYRP1 bypassed the need for peptidoglycan in TREM-1 activation, demonstrating that the PGLYRP1/TREM-1 axis can be activated in the absence of bacterial products. The role for PGLYRP1 as a TREM-1 activator provides a new mechanism by which bacteria can trigger myeloid cells, linking two known, but previously unrelated, pathways in innate immunity.

Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2

Starting from total pancreatic mRNAs, the classical guinea pig pancreatic lipase was cloned using rapid amplification of 3 and 5 cDNA ends. Internal oligonucleotide primers were designed from a partial cDNA clone including the region coding for the lid domain. Using this strategy, we did not amplify the cDNA corresponding to the pancreatic lipase related protein 2 in which the lid domain is deleted. Amino acid sequences of the classical guinea pig pancreatic lipase and the related protein 2 were compared based on the primary and tertiary structures of the classical human pancreatic lipase. Their distinct physiological roles are discussed in the light of functional amino acid differences.

Typical Danish Caucasian type 2 diabetic patients do not commonly carry genetic variants in GIP and GLP-1 encoding regions of the proGIP and proglucagon genes

BACKGROUNDnThe enteroinsular-axis is abnormal in type 2 diabetics, which contributes to the diabetic phenotype. The effect of the incretin hormone gastric inhibitory polypeptide (GIP) and the secretion of the incretin hormone glucagon-like peptide-1 (GLP-1) are thus greatly diminished. The explanation for these changes could be changes in the structure of either of the hormones or their receptors. Thus, the aim of this study was to study the occurrence of genetic variants in the GIP and GLP-1 encoding regions of the proGIP and proglucagon genes in type 2 diabetic patients and matched control subjects.nnnMETHODS AND RESULTSnGenomic DNA was extracted from buffy coats from 12 Caucasian type 2 diabetics and 12 healthy subjects, matched with respect to sex, age and BMI. The GIP and GLP-1 sequences were amplified using specific primers using the polymerase chain reaction (PCR). The amplified products were then sequenced. No germ-line mutations were identified in the GIP and the GLP-1 encoding regions of the proGIP and proglucagon genes in either the type 2 diabetic or the control subjects.nnnCONCLUSIONSnThe perturbed incretin effect in type 2 diabetics is not commonly caused by genetic variants in either the GIP or the GLP-1 encoding genes in type 2 diabetics.

IL-21 Increased Potency Design

Publisher Summary This chapter focuses on cytokine variants which have distinguished themselves by exhibiting altered biological activity compared to the naturally occurring (wt) cytokine in terms of enhanced potency in biological assays. It also emphasizes the identification of super-agonists, which arise from the exchange of amino acid residues within the cytokine protein per se. The growth hormone (hGH) is a monomeric protein, and binds the hGH receptor in a 1:2 ratio via two non-identical sites, called sites 1 and 2. Variants of hGH exhibiting improved binding affinity to the hGH receptor have been identified through an extensive affinity maturation approach, and one such “super-binding” variant, hGH, encompassing a total of 15 single-substitutions, has been demonstrated to bind with a 400-fold enhanced affinity to the hGH receptor. A saturation mutagenesis employing a minimized yet fully active form of IL-3 has revealed that 1-2 percent out of a total of more than 700 individual single-site mutants exhibited enhanced activity, emphasizing that the naturally occurring cytokine represents but one active form of the protein molecule, albeit not the most active form. The cytokine IL-6 signals through a receptor complex composed of a selective chain, IL-6Rα, and a common chain, gp130, which is shared by other cytokine receptor complexes. The IL-6 receptor complex differs distinctly from the heterodimeric γC cytokine complexes in the sense that the gp130 chain by itself constitutes the signaling receptor subunit, while IL-6Rα solely provides affinity and even does so either in cis, when membrane-embedded along with gp130, or in trans, when present in solution or on another cell.