Methods for characterization of hyaluronan from biological samples

Hyaluronan (HA) in human milk mediates host responses to microbial infection, via TLR4- and CD44-dependent signaling. Signaling by HA is generally size-specific. Pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells. The triggered protective response via pure 35 kDa HA stimulation is TLR4- but not CD44-dependent. Moreover, milk HA is ~700 times more effective than pure 35 kDa HA on a mass basis in inducing comparable innate immune response. It is therefore hypothesized that human milk may contain a high level of low M HA around 35 kDa, together with other larger sizes to interact with CD44 to enhance the innate immune response. Characterization of HA from human milk is necessary in order to provide effective information to test the hypothesis and to elucidate the relevant biological processes. There are several challenges associated with the characterization of HA from human milk. Milk is a viii highly complex fluid, and the current established HA isolation procedures are insufficient for removal of all contaminants. The concentration of HA in milk is low, so that methods of high sensitivity are required for analysis of the isolated HA. To address the issues of limited sample amount, purification difficulty, and the importance of analyzing both high and low M HA simultaneously, it was therefore the focus of this project to develop improved and highly sensitive methods to accurately analyze the HA content from human milk.;Chapter 1 attempted to give out a brief summary of background knowledge of hyaluronan. The unique biophysical properties of HA, as well as the relevant several important physiological functions, such as extracellular matrix stabilization, joint lubrication, shock absorption, water maintenance, and regulation of protein distribution are described. The biological regulation part of HA is then emphasized. The synthesis, metabolism, wellstudied interactions with some proteins, and some of the HA related biological processes including atherosclerosis and cancer are introduced.;Chapter 2 described hyaluronan quantification by applying specific and sensitive surface detection means, including a sandwich ELSA (enzyme linked sorbent assay) and membrane blotting. The molecular mass dependent detection signal has been found in these detection systems. A molecular mass dependent signal correction factor was developed, and used to correct the signal intensity to obtain accurate HA quantification estimation in the sandwich ELSA. The correction method worked for HA with M between about 20 and 150 kDa, but lower M HA was too poorly detected for useful analysis. The physical basis of the M dependent detection was proposed to be the increase in detectoraccessible fraction of each surface-bound molecule as M increases.;Chapter 3 focused on developing a hyaluronan specific isolation method to overcome its isolation difficulty from biological samples for further characterization. A method to label a HA binding protein while maintaining its activity was successfully ix developed. The labeled HA binding protein has been applied to capture HA, and the coated magnetic beads were then used to specifically harvest HA from biological mixtures. Different HA binding proteins have also been tested for the isolation method. The HA recovery via this specific isolation method was almost 100 % for size down to ~20 kDa, but had a preferential loss for smaller size.;Chapter 4 concentrated on developing a sensitive method to separate and quantify low M HA (less than about 100 kDa) without thorough sample purification by applying anion exchange (IEX) chromatography and a competitive ELSA. The ion exchange (IEX) chromatographic process was shown to provide size-dependent separation of low M HA, and the ELSA quantified the HA in each fraction. The average M and range of M in each fraction was controlled by varying the NaCl step concentrations, and relationships between salt concentrations for elution and the M distribution of each HA fraction was established. It was showed that the IEX process does not cause preferential loss of any size HA, and the M distribution obtained by electrophoresis of the unfractionated sample was well matched by HA-specific assay of the IEX fractions. The IEX-ELISA method was applied to the characterization of the M distribution of HA in a human milk sample.;In chapter 5, the IEX-competitive ELSA characterization method was applied to analyze HA in human milk samples from twenty unique donors, and the results were reported. When separated into four fractions, milk HA with M 20 kDa, M 20-60 kDa, and M ≈ 60-110 kDa comprised an average of 1.5%, 1.4% and 2% of the total HA, respectively. The remaining 95% was HA with M 110 kDa. Electrophoretic analysis of the higher M HA from thirteen samples showed nearly identical M distributions, with an average M of ~440 kDa. This higher M HA component in human milk was proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low M HA components. In addition, the similar higher M HA molecular mass distributions among different human milk samples x may reflect a strictly regulated balance between HA synthesis and metabolism during lactation.;Chapter 6 proposed some ideas about further improving the IEX-ELSA for HA characterization, including further optimizing the separation process and elevating the detection sensitivity. The further improved separation may be developed via automated, redesigned, and simplified fractionation processes. The detection sensitivity can be enhanced through readjusting the detection range of current colorimetric assay, applying time resolved fluorescence measurement, and luminescent detection as well. The ELSA process may be further simplified via assay development from the concept of homogeneous time resolved fluorescence resonance energy transfer.