Paul Cloos

Post-Translational Modifications of Proteins: Implications for Aging, Antigen Recognition, and Autoimmunity

Proteins are complex organic molecules susceptible to numerous post-translational modifications occurring spontaneously during aging or as a consequence of physiologic or pathologic processes. Antigenicity and interactions of proteins with components of the immune system may be profoundly affected by post-translational modifications. Thus, modified self-antigens may be absent (not-tolerated) during early T-cell selection and trigger reactions by the immune system as they arise later in life. In turn, this may play a role in the initiation and pathogenesis of autoimmune diseases. This Review article presents an overview of protein modifications that have been shown to affect antigenicity and presentation of protein antigens in autoimmune diseases. The relevance of these observations is discussed, and the implications for future prophylactic and therapeutic interventions are outlined.

Type I Collagen Racemization and Isomerization and the Risk of Fracture in Postmenopausal Women: The OFELY Prospective Study

The Asp1211 residue of the1209AHDGGR1214 sequence of the C‐terminal cross‐linking telopeptide of type I collagen (CTX) can undergo spontaneous post‐translational modifications, namely, racemization and isomerization, which result in the formation of four isomers: the native form (α‐L) and three age‐related forms, that is, an isomerized form (β‐L), a racemized form (α‐D), and an isomerized/racemized (β‐D) form. Previous studies have suggested that changes in the pattern of type I collagen racemization/isomerization, which can be assessed in vivo by measuring the degradation products of the CTX isoforms, may be associated with alterations of bone structure. The aim of this study was to examine prospectively the value of the different urinary CTX isoforms and their related ratio in the prediction of osteoporotic fractures in 408 healthy untreated postmenopausal women aged 50‐89 years (mean, 64 years) who were part of the OFELY cohort. During a median 6.8 years follow‐up, 16 incident vertebral fractures and 55 peripheral fractures were recorded in 65 women. The baseline levels of the four CTX isoforms in women who subsequently had a fracture were compared with those of the 343 women who did not fracture. At baseline, women with fractures had increased levels of ratios of native α‐L‐CTX to age‐related isoforms (β‐L, α‐D, and β‐D) compared with controls (p < 0.01). In logistic regression analysis after adjustment for age, prevalent fractures, and physical activity, women with levels of α‐L/β‐L, α‐L/α‐D, and α‐L/β‐D‐CTX ratios in the highest quartile had a 1.5‐ to 2‐fold increased risk of fractures compared with women with levels in the three lowest quartiles with relative risk (RR) and 95% CI of 2.0 (1.2‐3.5), 1.8 (1.02‐2.7), and 1.5 (0.9‐2.7), respectively. Adjustment of α‐L/β‐L and α‐L/α‐D‐CTX ratios by the level of bone turnover assessed by serum bone alkaline phosphatase (ALP)‐ or femoral neck bone mineral density (BMD) decreased slightly the RR, which remained significant for the α‐L/β‐L‐CTX ratio (RR [95%] CI, 1.8 [1.1‐3.2] after adjustment for bone ALP, 1.8 [1.03‐3.1] after adjustment for BMD, and 1.7 [0.95‐2.9] after adjustment for both bone ALP and BMD). Women with both high α‐L/β‐L‐CTX ratio and high bone ALP had a 50% higher risk of fracture than women with either one of these two risk factors. Similarly, women with both increased CTX ratio and low femoral neck BMD (T score < −2.5) had a higher risk of fracture with an RR (95% CI) of 4.5 (2.0‐10.1). In conclusion, increased urinary ratio between native and age‐related forms of CTX, reflecting decreased degree of type I collagen racemization/isomerization, is associated with increased fracture risk independently of BMD and partly of bone turnover rate. This suggests that alterations of type I collagen isomerization/racemization that can be detected by changes in urinary CTX ratios may be associated with increased skeletal fragility.

Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications

Given the complexity of the biosynthetic machinery and the delicate chemical composition of proteins, it is remarkable that cells manage to produce and maintain normally functioning proteins under most conditions. However, it is now well known that proteins are susceptible to various non-enzymatic covalent modifications (NECM) under physiological conditions. Such modifications can be of no or little importance to the protein or they can be absolutely detrimental. Often NECM are difficult to study due to the complex and technically demanding methods required to identify many of these modifications. Thus, the role of NECM has not yet been adequately resolved but recent research has allowed a better understanding of such modifications. The present review outlines the various forms of NECM that involve covalent modifications of proteins, and discusses their relevance, biological impact and potential applications in the study of protein turnover and diagnosis of disease.

Non‐Isomerized C‐Telopeptide Fragments Are Highly Sensitive Markers for Monitoring Disease Activity and Treatment Efficacy in Paget's Disease of Bone

A new resorption assay measuring non‐isomerized collagen type I C‐telopeptide fragments (α‐α CTX) was evaluated in a cohort comprising 32 Pagetic patients and 48 healthy controls. α‐α CTX was found to be a sensitive marker for assessing disease activity and monitoring treatment efficacy in Pagets disease of bone compared with isomerized CTX (β‐β CTX) and a number of other established bone turnover markers.

Investigation of bone disease using isomerized and racemized fragments of type I collagen.

In the collagen type I C-telopeptide an aspartyl-glycine site within the sequence AHDGGR is susceptible to molecular rearrangement. In newly synthesized collagen this site is in the native form, denoted aL. During aging a spontaneous reaction occurs resulting in three age-modified forms: an isomerized form (bL) a racemized form (aD), and an isomerized/racemized form (bD). In this study, we measured the urinary excretion of the four forms of C-telopeptides (CTX) in healthy adults and in patients with bone diseases. Levels of all CTX forms were higher in healthy postmenopausal women (P<0.001) compared with premenopausal controls. Levels decreased within 3 days of bisphosphonate treatment indicating that all CTX forms reflect bone resorption. In hyperthyroidism, characterized by a generalized increased bone turnover, native (aL) and age-modified (bL, aD and bD) forms increased to a similar extent compared to controls, resulting in normal ratios between the aL and age-modified forms of CTX. Conversely, in Pagets disease and prostate cancer-induced bone metastases, conditions characterized by focal increased bone turnover, aL CTX levels were more elevated than those of age-related CTX forms, resulting in increased ratios between native and age-modified CTX. For example, the ratio aL/aD was increased 7-fold in Pagets disease (P<0.001) and 2-fold in prostate cancer-induced bone metastases (P<0.002). In conclusion, the study suggests that in conditions with a localized alteration in bone turnover the ratio between aL CTX and the age-modified forms is significantly elevated. This may provide a new diagnostic and monitoring tool for diseases such as metastatic bone cancer and Pagets disease.

Breast cancer patients with bone metastases are characterised by increased levels of nonisomerised type I collagen fragments

BackgroundFragments of collagen type I containing the epitope AHDGGR (CTX) are generated during bone resorption. The aspartyl-glycine (DG) site within CTX is synthesised in the L-aspartyl peptide (αL) form, but converts to the age-modified forms L-isoaspartyl peptide (βL) and D-aspartyl peptide (αD) over time. The purpose of the present study was to test the ability of the various CTX forms to identify breast cancer patients with bone metastases and to investigate whether such patients had an altered CTX excretion pattern.MethodsIn this cross-sectional study we compared CTX excretion in healthy premenopausal and postmenopausal women with CTX levels in patients with breast cancer. The breast cancer cohort comprised eight hypercalcemic patients with bone metastases (HC+), 100 normocalcemic patients with bone metastases (NC+) and 15 normocalcemic patients without bone metastases (NC-).ResultsIn HC+ patients and NC+ patients, the excretion of αL CTX was highly increased compared with NC- patients (P < 0.01), with Z scores of 3.4 and 2.0, respectively. The excretion of the age-modified forms (βL and αD CTX) was less increased in HC+ patients and in NC+ patients as compared with NC- patients, with Z scores of 2.2 and 1.0, respectively, for βL CTX, and of 1.6 and 0.8, respectively, for αD CTX.ConclusionAssays for the various isoforms of CTX have different sensitivities to identify patients affected by bone metastases. The αL CTX isoform reflecting resorption of young bone appeared to provide the best differentiation of patients affected by breast cancer-induced bone metastases. In conclusion, patients affected by metastatic bone disease present an altered excretion pattern of CTX isoforms.

Monoclonal antibodies to degradation products of type I collagen reflecting bone resorption

The specific bone marker Osteocalcin is prone to rapid degradation at the 6 amino acids of the C-terminal. Thus in order to get reliable estimates of the seru~ osteocalcin level, an assay independent of the labile Cterminal is needed. Here we present a new immuno radio~etric assay based on two monoclonal antibodies, one specific for amino acid 7-19 and one specific for amino acid 20-43. The method is a simple convenient two step procedure with a total duration of 2 hrs. Osteocalcin ranging from 0.5 ng/mL to 130 ng/mL was measured with low imprecision: 6.8 % between run and 6.4 % within run. Samples were readily diluted with assay buffer or spiked with synthetic osteocalcin yielding recoveries near 100 %, which excluded the possible serum matrix effects. Normal ranges of hOsteocalcin was established by analysing sera from healthy subjects. The level was 18.4 • 8.9 ng/mL in healthy premenopausal women (n = 47) and 29.9 • 11.5 ng/mL in healthy postmenopausal women (n = 100). A high correlation was seen to N MID TM ELISA (Osteometer BioTech A/S, Herlev, Denmark). The N-MID TM IRMA showed great potency for evaluation of anti-resorptive treatments. Premenopausal women, who were treated with oestrogen therapy showed significant decrease in the serum osteocalcin level as measured by the IRMA. Osteocalcin in the active group (n=10) decreased to 75 % of the level at time of inclusion, whereas the level in the placebo group increased to 115 %. Bisphosphonate treatment was likewise monitored by the N-MID TM IRMA. Highest decrease of serum osteocalcin was seen in the patient group receiving active treatment, that is to 39.8 %, whereas the placebo group only decreased to 66 %. Patients were also receiving calcium throughout the entire study. In conclusion, the developed N-MID IRMA will be a valuable tool for monitoring anti-resorptive therapy.

Specificity of the crosslapsTM ELISA and the MAbA7 ELISA

The specific bone marker Osteocalcin is prone to rapid degradation at the 6 amino acids of the C-terminal. Thus in order to get reliable estimates of the seru~ osteocalcin level, an assay independent of the labile Cterminal is needed. Here we present a new immuno radio~etric assay based on two monoclonal antibodies, one specific for amino acid 7-19 and one specific for amino acid 20-43. The method is a simple convenient two step procedure with a total duration of 2 hrs. Osteocalcin ranging from 0.5 ng/mL to 130 ng/mL was measured with low imprecision: 6.8 % between run and 6.4 % within run. Samples were readily diluted with assay buffer or spiked with synthetic osteocalcin yielding recoveries near 100 %, which excluded the possible serum matrix effects. Normal ranges of hOsteocalcin was established by analysing sera from healthy subjects. The level was 18.4 • 8.9 ng/mL in healthy premenopausal women (n = 47) and 29.9 • 11.5 ng/mL in healthy postmenopausal women (n = 100). A high correlation was seen to N MID TM ELISA (Osteometer BioTech A/S, Herlev, Denmark). The N-MID TM IRMA showed great potency for evaluation of anti-resorptive treatments. Premenopausal women, who were treated with oestrogen therapy showed significant decrease in the serum osteocalcin level as measured by the IRMA. Osteocalcin in the active group (n=10) decreased to 75 % of the level at time of inclusion, whereas the level in the placebo group increased to 115 %. Bisphosphonate treatment was likewise monitored by the N-MID TM IRMA. Highest decrease of serum osteocalcin was seen in the patient group receiving active treatment, that is to 39.8 %, whereas the placebo group only decreased to 66 %. Patients were also receiving calcium throughout the entire study. In conclusion, the developed N-MID IRMA will be a valuable tool for monitoring anti-resorptive therapy.

Application of a monoclonal antibody for measurement of bone collagen derived peptide pragments in urine

The CrossLaps TM ELISA employs a polyclonal rabbit antibody for measurement of bone collagen derived peptide fragments in urine The molecules measured in this assay have been shown to reflect bone resorption (P. Garnero et el, 1994, J Clin Endocrinol Metab, 79, 78D-785). We have recently found that the collagen derived octapeptlde (EK~.HDGGR), recognized in the CrossLaps ~ ELISA, is subject to isomerization at the aspartate residue resulting in a ~-aspartate linkage (EKAHD-~-GGR), and only the isomerized forms of the epitope are recognized in the assay. Here, we report the production of a monoclonal antibody i6E) reactive with the same isomerized ocfapeptide segment from the C-terminal telopeptide of type I collagen, and we describe the development of immunoassays with the same specificity as the CrossLaps ~ ELISA, but based on the monoclonal antibody, The 6E antibody recognized both a hexapeptide (~/4D-J-GGR} and to a lesser extent a tetrapeptide (D-~-GGR) containing the C terminal segment of the epitope used for immunization. However, the antibody was u~able to bind a septapeptide lacking the C-terminal arginine residue (EKAHD-~-GG) and a nonapeptide prolonged with one amino acid C-terminally (DK~D-~-GGRY), demonstrating the importance of the C-terminal free arginine for binding of the antibody. Two assay formats employing the 6E antibody were developed, a competitive ELISA assay and a radio-immunoassay (RIA), Urine samples were measured in both assays and the results were compared to measurements obtained with the urine CrossLaps ~ ELISA. The ELISA assay showed correlation of 0.91 and the RIA assay showed a correlation of 0.92 to the urine CrossLaps TM ELISA, suggesting that the 6E assays measure a highly similar population of collagen fragments in urine as the CrossLaps TM ELISA. We evaluated the ability of the 6E ELISA to measure the decreased bone resorption resulting from bisphosphonate therapy Urine samples were obtained from a double blind, placebo controlled, study of the effect of Ibandronate TM treatment in postmenopausal women with low bone mass. Samples were taken at baseline, and after three months from 20 women treated with 2.5 mg Ibandronate TM daily, and 20 women in a placebo group given only calcium. The samples were measured in the CrossLaps TM ELISA and in the 6E ELISA. In both assays a decrease of more than 70 % were observed in the Ibandronate TM treated group, whereas the placebo group did not change significantly These results strongly suggest that assays employing the 6E antibody will have the same clinical performance as the CrossLaps ~ ELISA and that such immunoassays have a high clinical relevance for monitoring anti resorptive therapy in osteoporosis and other diseases affecting bone metabolism.