Jean L. Spencer

Heparan Sulfate-Protein Binding Specificity

Heparan sulfate (HS) represents a large class of linear polysaccharides that are required for the function of all mammalian physiological systems. HS is characterized by a repeating disaccharide backbone that is subject to a wide range of modifications, making this class of macromolecules arguably the most information dense in all of biology. The majority of HS functions are associated with the ability to bind and regulate a wide range of proteins. Indeed, recent years have seen an explosion in the discovery of new activities for HS where it is now recognized that this class of glycans functions as co-receptors for growth factors and cytokines, modulates cellular uptake of lipoproteins, regulates protease activity, is critical to amyloid plaque formation, is used by opportunistic pathogens to enter cells, and may even participate in epigenetic regulation. This review will discuss the current state of understanding regarding the specificity of HS-protein binding and will describe the concept that protein binding to HS depends on the overall organization of domains within HS rather than fine structure.

A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema

Two million Americans suffer from pulmonary emphysema, costing

A Computational Approach for Deciphering the Organization of Glycosaminoglycans

2.5 billion/year and contributing to 100,000 deaths/year. Emphysema is thought to result from an imbalance between elastase and endogenous inhibitors of elastase, leading to tissue destruction and a loss of alveoli. Decades of research have still not resulted in an effective treatment other than stopping cigarette smoking, a highly addictive behavior. On the basis of our previous work, we hypothesize that small molecule inhibitors of human neutrophil elastase are ineffective because of rapid clearance from the lungs. To develop a long-acting elastase inhibitor with a lung pharmacodynamic profile that has minimal immunogenicity, we covalently linked an elastase inhibitor, similar to a trifluoro inhibitor that was used in clinical trials, to a 25-amino-acid fragment of human surfactant peptide B. We used this construct to prevent human neutrophil elastase-induced emphysema in a rodent model. The elastase inhibitor alone, although in a 70-fold molar excess to elastase in a mixture with <0.6% residual elastase activity, provided no protection from elastase-induced emphysema. Covalently combining an endogenous peptide from the target organ with a synthetic small molecule inhibitor is a unique way of endowing an active compound with the pharmacodynamic profile needed to create in vivo efficacy.

Dynamics of enzymatic digestion of elastic fibers and networks under tension.

Background Increasing evidence has revealed important roles for complex glycans as mediators of normal and pathological processes. Glycosaminoglycans are a class of glycans that bind and regulate the function of a wide array of proteins at the cell-extracellular matrix interface. The specific sequence and chemical organization of these polymers likely define function; however, identification of the structure-function relationships of glycosaminoglycans has been met with challenges associated with the unique level of complexity and the nontemplate-driven biosynthesis of these biopolymers. Methodology/Principal Findings To address these challenges, we have devised a computational approach to predict fine structure and patterns of domain organization of the specific glycosaminoglycan, heparan sulfate (HS). Using chemical composition data obtained after complete and partial digestion of mixtures of HS chains with specific degradative enzymes, the computational analysis produces populations of theoretical HS chains with structures that meet both biosynthesis and enzyme degradation rules. The model performs these operations through a modular format consisting of input/output sections and three routines called chainmaker, chainbreaker, and chainsorter. We applied this methodology to analyze HS preparations isolated from pulmonary fibroblasts and epithelial cells. Significant differences in the general organization of these two HS preparations were observed, with HS from epithelial cells having a greater frequency of highly sulfated domains. Epithelial HS also showed a higher density of specific HS domains that have been associated with inhibition of neutrophil elastase. Experimental analysis of elastase inhibition was consistent with the model predictions and demonstrated that HS from epithelial cells had greater inhibitory activity than HS from fibroblasts. Conclusions/Significance This model establishes the conceptual framework for a new class of computational tools to use to assess patterns of domain organization within glycosaminoglycans. These tools will provide a means to consider high-level chain organization in deciphering the structure-function relationships of polysaccharides in biology.

STRAP PTM: Software Tool for Rapid Annotation and Differential Comparison of Protein Post‐Translational Modifications

We study the enzymatic degradation of an elastic fiber under tension using an anisotropic random-walk model coupled with binding-unbinding reactions that weaken the fiber. The fiber is represented by a chain of elastic springs in series along which enzyme molecules can diffuse. Numerical simulations show that the fiber stiffness decreases exponentially with two distinct regimes. The time constant of the first regime decreases with increasing tension. Using a mean field calculation, we partition the time constant into geometrical, chemical and externally controllable factors, which is corroborated by the simulations. We incorporate the fiber model into a multiscale network model of the extracellular matrix and find that network effects do not mask the exponential decay of stiffness at the fiber level. To test these predictions, we measure the force relaxation of elastin sheets stretched to 20% uniaxial strain in the presence of elastase. The decay of force is exponential and the time constant is proportional to the inverse of enzyme concentration in agreement with model predictions. Furthermore, the fragment mass released into the bath during digestion is linearly related to enzyme concentration that is also borne out in the model. We conclude that in the complex extracellular matrix, feedback between the local rate of fiber digestion and the force the fiber carries acts to attenuate any spatial heterogeneity of digestion such that molecular processes manifest directly at the macroscale. Our findings can help better understand remodeling processes during development or in disease in which enzyme concentrations and/or mechanical forces become abnormal.

Experimental observations of gas phase–adsorbed phase interactions during counterdiffusion in porous alumina

The identification of protein post‐translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography–tandem mass spectrometry experiments. STRAP PTM (Software Tool for Rapid Annotation of Proteins: Post‐Translational Modification edition) is a program that was developed to facilitate the characterization of PTMs using spectral counting and a novel scoring algorithm to accelerate the identification of differential PTMs from complex data sets. The software facilitates multi‐sample comparison by collating, scoring, and ranking PTMs, and by summarizing data visually. The freely available software (beta release) installs on a PC and processes data in protXML format obtained from files parsed through the Trans‐Proteomic Pipeline. The easy‐to‐use interface allows examination of results at protein, peptide, and PTM levels, and the overall design offers tremendous flexibility that provides proteomics insight beyond simple assignment and counting. Curr. Protoc. Bioinform. 44:13.22.1‐13.22.36.

New insights into the inhibition of human neutrophil elastase by heparin.

The binary gas systems helium–argon, helium–nitrogen, helium–1‐butene, and argon–1‐butene were counterdiffused through a plug of porous alumina at 135° and 150 °C and 1–3 atm pressure in a Wicke–Kallenbach experiment. The helium, nitrogen, and argon were essentially nonadsorbing; the butene was significantly adsorbed and diffused along the surface. Knudsen flow existed within the pores at most experimental conditions, i.e., the gas–gas molecular collisions were negligible in comparison with gas–surface collisions. The results showed that gas‐adsorbed phase collisions can have significant effects at conditions typical of laboratory and industrial practice. Strong evidence of interactions between all gas‐phase molecules and adsorbed butene molecules was observed whenever the butene was present. Both helium and argon fluxes were significantly less when counterdiffused with 1‐butene than when counterdiffused with nonadsorbing gases. The butene flux was markedly less when diffused against the argon than when d...